JP2016539946A - MERS-CoV vaccine - Google Patents

Abstract

Disclosed herein is a vaccine comprising the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) antigen. The antigen can be a consensus antigen. The consensus antigen can be a consensus spike antigen. A method of treating a patient in need of treatment by administering the vaccine to the patient is also disclosed. [Selection] Figure 1

Description

TECHNICAL FIELD The present invention relates to a vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) and a method for administering the vaccine.

CROSS REFERENCE TO RELATED APPLICATIONS This application is a U.S. application filed November 29, 2013. S. Provisional Patent Application No. The priority of 61 / 910,153 is claimed and incorporated herein by reference.

Background Coronavirus (CoV) is a type of virus that is common throughout the world and causes human diseases ranging from the common cold to severe acute respiratory syndrome (SARS). Coronaviruses can cause many diseases in animals. Human coronavirus 229E, OC43, NL63, and HKU1 are unique to the human population.

  In 2012, a new type of coronavirus (nCoV) appeared in Saudi Arabia and is now known as the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) (FIG. 1). MERS-CoV can be classified as a beta coronavirus (FIG. 2, MERS-CoV strain HCoV-EMC / 2012 with an asterisk). Subsequent cases of MERS-CoV infection have been reported throughout the Middle East (eg, Qatar and Jordan) and more recently in Europe. MERS-CoV infection manifested itself as a severe acute respiratory disease with symptoms of fever, cough, and shortness of breath. About half of the reported cases of MERS-CoV infections were fatal, with the majority of reported cases from older to middle-aged men. Only a few of the reported cases included patients with mild respiratory disease. Infections from person to person with MERS-CoV can occur, but at present are very limited.

  Therefore, there remains a need in the art to develop a safe and effective vaccine that can be applied to MERS-CoV, thereby preventing MERS-CoV infection and promoting its survival.

  The present invention relates to immunogenic compositions. In one embodiment, the invention is a vaccine comprising a nucleic acid molecule, wherein the nucleic acid molecule can comprise a nucleic acid sequence that is at least about 90% homologous to the full length of the nucleic acid sequence set forth in SEQ ID NO: 1, or the nucleic acid The vaccine relates to a vaccine wherein the molecule may comprise a nucleic acid sequence that is at least about 90% homologous to the full length of the nucleic acid sequence set forth in SEQ ID NO: 3.

  The present invention is a vaccine comprising a nucleic acid molecule, wherein the nucleic acid molecule can encode a peptide comprising an amino acid sequence that is at least about 90% homologous to the full length of the amino acid sequence set forth in SEQ ID NO: 2, or the nucleic acid molecule Also relates to a vaccine capable of encoding a peptide comprising an amino acid sequence that is at least about 90% homologous to the full length of the amino acid sequence set forth in SEQ ID NO: 4.

  The present invention further relates to a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 1.

  The present invention further relates to a nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 3.

  The present invention further relates to a peptide comprising the amino acid sequence set forth in SEQ ID NO: 2.

  The present invention further relates to a peptide comprising the amino acid sequence set forth in SEQ ID NO: 4.

  The present invention further relates to a vaccine comprising an antigen, wherein the antigen is encoded by SEQ ID NO: 1 or SEQ ID NO: 3.

  The present invention is a vaccine comprising a peptide, wherein the peptide may comprise an amino acid sequence that is at least about 90% homologous to the full length of the amino acid sequence set forth in SEQ ID NO: 2, or the peptide is set forth in SEQ ID NO: 4 Also relates to a vaccine that may comprise an amino acid sequence that is at least about 90% homologous to the full length of

  The invention further relates to a method of inducing an immune response against a Middle East respiratory syndrome coronavirus (MERS-CoV) in a patient in need thereof. The method can include administration of one or more of the vaccines, nucleic acid molecules, or peptides to the patient.

  The invention further relates to a method for protecting patients in need of protection from infection with the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The method can include administration of one or more of the vaccines, nucleic acid molecules, or peptides to the patient.

  The invention further relates to a method for treating a patient in need of treatment for the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The method can include administration of one or more of the vaccines, nucleic acid molecules, or peptides to the patient.

A time series explaining the appearance of MERS-CoV is shown. A phylogenetic tree depicting the relationship between the indicated coronaviruses is shown. FIG. 2 shows a phylogenetic tree depicting the relationship between the shown MERS-CoV and MERS-CoV consensus spike antigens. (A) A schematic representation of the MERS-HCoV-WT construct is shown. (B) An image of the stained gel is shown. Fig. 4 shows a graph created by the web software Phobius. The figure made by web software InterProScan is shown. (A) Schematic representation of the MERS-HCoV-ΔCD construct is shown. (B) An image of the stained gel is shown. (A) shows the nucleotide sequence encoding the MERS-CoV consensus spike antigen. (A) shows the nucleotide sequence encoding the MERS-CoV consensus spike antigen. (B) shows the amino acid sequence of MERS-CoV consensus spike antigen. (C) shows the nucleotide sequence encoding the MERS-CoV consensus spike antigen lacking the cytoplasmic domain (MERS-CoV consensus spike antigen ΔCD). (C) shows the nucleotide sequence encoding the MERS-CoV consensus spike antigen lacking the cytoplasmic domain (MERS-CoV consensus spike antigen ΔCD). (D) shows the amino acid sequence of MERS-CoV consensus spike antigen ΔCD. Immunoblots are shown. A schematic representation of the immunization regimen is shown. The graph which plotted blood collection and OD450 is shown to (A) and (B). The graph which plotted blood collection and OD450 is shown to (A) and (B). A matrix of peptide pools is shown. The graph which plotted the peptide pool and the spot formation unit per 10 ⁶ cells (SFU / 10 ⁶ cells) is shown. The graph which plotted the peptide pool and the spot formation unit per 10 ⁶ cells (SFU / 10 ⁶ cells) is shown. The graph which plotted the immunization group and the spot formation unit per 10 ⁶ cells (SFU / 10 ⁶ cells) is shown. (A) Graph plotting immunization group and CD3 ⁺ CD4 ⁺ IFN-γ ⁺ T cell%; (B) Graph plotting immunization group and CD3 ⁺ CD4 ⁺ TNF-α ⁺ T cell%; (C) Immunization The graph which plotted the immunization group and CD3 ⁺ CD4 ⁺ IL-2 ⁺ T cell%; and (D) The graph which plotted the immunization group and CD3 ⁺ CD4 ⁺ IFN-γ ⁺ TNF-α ⁺ T cell%. (A) Graph plotting immunization group and CD3 ⁺ CD8 ⁺ IFN-γ ⁺ T cell%; (B) Graph plotting immunization group and CD3 ⁺ CD8 ⁺ TNF-α ⁺ T cell%; (C) Immunization The graph which plotted the immunization group and CD3 ⁺ CD8 ⁺ IL-2 ⁺ T cell%; and (D) The graph which plotted the immunization group and CD3 ⁺ CD8 ⁺ IFN-γ ⁺ TNF-α ⁺ T cell%. (A) shows antibody responses after vaccination with pVax1 DNA vector (negative control), consensus MERS-Spike DNA vaccine (MERS-S), or consensus MERS-Spike-ΔCD DNA vaccine (MERS-S-CD). Show. The antibody reaction was measured by ELISA. (B) shows antibody response over time after vaccination with consensus MERS-Spike DNA vaccine (MERS-S). (C) shows neutralizing antibody titers 35 days after vaccination with pVax1 DNA vector (negative control), MERS-S DNA vaccine, or MERS-S-CD DNA vaccine. Mouse serum was serially diluted with MEM and cultured at 37 ° C. with 50 ul of DMEM containing 100 infectious HCoV-EMC / 2012 (Human Coronavirus Erasmus Medical Center / 2012) per well. After 90 minutes, the virus-serum mixture was added to Vero cell monolayers (100,000 cells / well) in 96-well flat bottom plates and cultured at 37 ° C. in a 5% CO ₂ incubator for 5 days. The neutralizing antibody titer for each specimen is shown as the maximum dilution at which less than 50% of the cells show CPE. Values are given as reciprocal dilutions. All samples were tested in duplicate and the final result was the average of the two values. The neutralization rate was calculated as follows: Neutralization rate = {1-PFU of target mAb (each concentration) / average PFU of negative control (total concentration)}. (A) shows antibody responses after vaccination with pVax1 DNA vector (negative control), consensus MERS-Spike DNA vaccine (MERS-S), or consensus MERS-Spike-ΔCD DNA vaccine (MERS-S-CD). Show. The antibody reaction was measured by ELISA. (B) shows antibody response over time after vaccination with consensus MERS-Spike DNA vaccine (MERS-S). (C) shows neutralizing antibody titers 35 days after vaccination with pVax1 DNA vector (negative control), MERS-S DNA vaccine, or MERS-S-CD DNA vaccine. Mouse serum was serially diluted with MEM and cultured at 37 ° C. with 50 ul of DMEM containing 100 infectious HCoV-EMC / 2012 (Human Coronavirus Erasmus Medical Center / 2012) per well. After 90 minutes, the virus-serum mixture was added to Vero cell monolayers (100,000 cells / well) in 96-well flat bottom plates and cultured at 37 ° C. in a 5% CO ₂ incubator for 5 days. The neutralizing antibody titer for each specimen is shown as the maximum dilution at which less than 50% of the cells show CPE. Values are given as reciprocal dilutions. All samples were tested in duplicate and the final result was the average of the two values. The neutralization rate was calculated as follows: Neutralization rate = {1-PFU of target mAb (each concentration) / average PFU of negative control (total concentration)}. (A) shows antibody responses after vaccination with pVax1 DNA vector (negative control), consensus MERS-Spike DNA vaccine (MERS-S), or consensus MERS-Spike-ΔCD DNA vaccine (MERS-S-CD). Show. The antibody reaction was measured by ELISA. (B) shows antibody response over time after vaccination with consensus MERS-Spike DNA vaccine (MERS-S). (C) shows neutralizing antibody titers 35 days after vaccination with pVax1 DNA vector (negative control), MERS-S DNA vaccine, or MERS-S-CD DNA vaccine. Mouse serum was serially diluted with MEM and cultured at 37 ° C. with 50 ul of DMEM containing 100 infectious HCoV-EMC / 2012 (Human Coronavirus Erasmus Medical Center / 2012) per well. After 90 minutes, the virus-serum mixture was added to Vero cell monolayers (100,000 cells / well) in 96-well flat bottom plates and cultured at 37 ° C. in a 5% CO ₂ incubator for 5 days. The neutralizing antibody titer for each specimen is shown as the maximum dilution at which less than 50% of the cells show CPE. Values are given as reciprocal dilutions. All samples were tested in duplicate and the final result was the average of the two values. The neutralization rate was calculated as follows: Neutralization rate = {1-PFU of target mAb (each concentration) / average PFU of negative control (total concentration)}. The structure and characteristic of MERS-HCoV Spike DNA vaccine are shown. (A) Phylogenetic tree showing the phylogenetic relationship at the amino acid level of MERS-HCoV virus isolates and Spike vaccine strains. The rooted neighbor-joining tree arises from the amino acid sequence alignment of the Spike protein of the consensus vaccine indicated by (*), and (♦) indicates the MERS virus strain used in the Nab analysis test. The shaded area represents reported cases from the 2012-2013 outbreak. Specific branch groups are shown in parentheses. The tree was drawn at a constant scale (scale bar, 0.1% difference). (B) Schematic representation of Spike-Wt gene insert used in codon optimized DNA vaccine. Spike-Wt and SpikeΔCD-DNA vaccines were constructed. Different Spike protein domains were shown. (C) Protein expression was detected by Western blot. The expression of the Spike-Wt and SpikeΔCD-Spike proteins was analyzed 2 days after transfection by Western blot with a 1: 100 dilution using sera from MERS-Spike-Wt DNA vaccinated mice. The transfected DNA construct and the amount of cell lysate loaded are as indicated. The spike protein is indicated by an arrow. (D) Immunofluorescence analysis in Vero cells showing Spike-Wt and SpikeΔCD protein expression using serum (1: 100) from mice immunized with DNA. The structure and characteristic of MERS-HCoV Spike DNA vaccine are shown. (A) Phylogenetic tree showing the phylogenetic relationship at the amino acid level of MERS-HCoV virus isolates and Spike vaccine strains. The rooted neighbor-joining tree arises from the amino acid sequence alignment of the Spike protein of the consensus vaccine indicated by (*), and (♦) indicates the MERS virus strain used in the Nab analysis test. The shaded area represents reported cases from the 2012-2013 outbreak. Specific branch groups are shown in parentheses. The tree was drawn at a constant scale (scale bar, 0.1% difference). (B) Schematic representation of Spike-Wt gene insert used in codon optimized DNA vaccine. Spike-Wt and SpikeΔCD-DNA vaccines were constructed. Different Spike protein domains were shown. (C) Protein expression was detected by Western blot. The expression of the Spike-Wt and SpikeΔCD-Spike proteins was analyzed 2 days after transfection by Western blot with a 1: 100 dilution using sera from MERS-Spike-Wt DNA vaccinated mice. The transfected DNA construct and the amount of cell lysate loaded are as indicated. The spike protein is indicated by an arrow. (D) Immunofluorescence analysis in Vero cells showing Spike-Wt and SpikeΔCD protein expression using serum (1: 100) from mice immunized with DNA. The structure and characteristic of MERS-HCoV Spike DNA vaccine are shown. (A) Phylogenetic tree showing the phylogenetic relationship at the amino acid level of MERS-HCoV virus isolates and Spike vaccine strains. The rooted neighbor-joining tree arises from the amino acid sequence alignment of the Spike protein of the consensus vaccine indicated by (*), and (♦) indicates the MERS virus strain used in the Nab analysis test. The shaded area represents reported cases from the 2012-2013 outbreak. Specific branch groups are shown in parentheses. The tree was drawn at a constant scale (scale bar, 0.1% difference). (B) Schematic representation of Spike-Wt gene insert used in codon optimized DNA vaccine. Spike-Wt and SpikeΔCD-DNA vaccines were constructed. Different Spike protein domains were shown. (C) Protein expression was detected by Western blot. The expression of the Spike-Wt and SpikeΔCD-Spike proteins was analyzed 2 days after transfection by Western blot with a 1: 100 dilution using sera from MERS-Spike-Wt DNA vaccinated mice. The transfected DNA construct and the amount of cell lysate loaded are as indicated. The spike protein is indicated by an arrow. (D) Immunofluorescence analysis in Vero cells showing Spike-Wt and SpikeΔCD protein expression using serum (1: 100) from mice immunized with DNA. The structure and characteristic of MERS-HCoV Spike DNA vaccine are shown. (A) Phylogenetic tree showing the phylogenetic relationship at the amino acid level of MERS-HCoV virus isolates and Spike vaccine strains. The rooted neighbor-joining tree arises from the amino acid sequence alignment of the Spike protein of the consensus vaccine indicated by (*), and (♦) indicates the MERS virus strain used in the Nab analysis test. The shaded area represents reported cases from the 2012-2013 outbreak. Specific branch groups are shown in parentheses. The tree was drawn at a constant scale (scale bar, 0.1% difference). (B) Schematic representation of Spike-Wt gene insert used in codon optimized DNA vaccine. Spike-Wt and SpikeΔCD-DNA vaccines were constructed. Different Spike protein domains were shown. (C) Protein expression was detected by Western blot. The expression of the Spike-Wt and SpikeΔCD-Spike proteins was analyzed 2 days after transfection by Western blot with a 1: 100 dilution using sera from MERS-Spike-Wt DNA vaccinated mice. The transfected DNA construct and the amount of cell lysate loaded are as indicated. The spike protein is indicated by an arrow. (D) Immunofluorescence analysis in Vero cells showing Spike-Wt and SpikeΔCD protein expression using serum (1: 100) from mice immunized with DNA. The structure and characteristics of MERS-Spike pseudovirus are shown. (A) Schematic representation of MERS-HCoV-Spike pseudovirus. (B) Quantification of viral infectivity: Vero cells were cultured with MERS-Spike pseudotyped with a luciferase reporter backbone. The luciferase reporter activity was then analyzed at the indicated times to obtain changes in expression over time. The curve matched the data from nonlinear regression analysis and the best fit was a straight line. (C) Detection of MERS-HCoV-Spike pseudovirus infectivity in different cell lines. MERS-Spike pseudovirus and negative control VSV-G virus were used for infection. Data were expressed as mean relative luciferase units (RLU) ± standard deviation (SD) of three parallel wells of a 96 well culture plate. The experiment was repeated three times with similar results. The structure and characteristics of MERS-Spike pseudovirus are shown. (A) Schematic representation of MERS-HCoV-Spike pseudovirus. (B) Quantification of viral infectivity: Vero cells were cultured with MERS-Spike pseudotyped with a luciferase reporter backbone. The luciferase reporter activity was then analyzed at the indicated times to obtain changes in expression over time. The curve matched the data from nonlinear regression analysis and the best fit was a straight line. (C) Detection of MERS-HCoV-Spike pseudovirus infectivity in different cell lines. MERS-Spike pseudovirus and negative control VSV-G virus were used for infection. Data were expressed as mean relative luciferase units (RLU) ± standard deviation (SD) of three parallel wells of a 96 well culture plate. The experiment was repeated three times with similar results. The structure and characteristics of MERS-Spike pseudovirus are shown. (A) Schematic representation of MERS-HCoV-Spike pseudovirus. (B) Quantification of viral infectivity: Vero cells were cultured with MERS-Spike pseudotyped with a luciferase reporter backbone. The luciferase reporter activity was then analyzed at the indicated times to obtain changes in expression over time. The curve matched the data from nonlinear regression analysis and the best fit was a straight line. (C) Detection of MERS-HCoV-Spike pseudovirus infectivity in different cell lines. MERS-Spike pseudovirus and negative control VSV-G virus were used for infection. Data were expressed as mean relative luciferase units (RLU) ± standard deviation (SD) of three parallel wells of a 96 well culture plate. The experiment was repeated three times with similar results. Figure 2 shows the functional profile of the cellular immune response elicited by MERS-HCoV vaccine. (A) As shown, C57 / BL6 mice group (n = 9 / group) were immunized 3 times with 2 weeks of Spike-Wt and SpikeΔCD DNA vaccine, respectively. Samples were collected one week after the third immunization. (B) Spike-specific T lymphocyte responses were assessed using IFN-γ ELISpot analysis using 6 peptide pools covering the Spike protein. Values represented the average response in each group one week after the third immunization. Standard error is shown in error bar. (C and D) Properties of MERS-Spike specific dominant epitope. Secretion of IFN-γ by splenocytes from MERS-Spike specific immune responses using matrix pooling of peptides. These results were expressed as the number of IFN-γ secreting cells / 10 ⁶ splenocytes after subtracting the value without peptide stimulation (mean ± SD of 3 mice per group). Similar values were obtained from two separate experiments. Figure 2 shows the functional profile of the cellular immune response elicited by MERS-HCoV vaccine. (A) As shown, C57 / BL6 mice group (n = 9 / group) were immunized 3 times with 2 weeks of Spike-Wt and SpikeΔCD DNA vaccine, respectively. Samples were collected one week after the third immunization. (B) Spike-specific T lymphocyte responses were assessed using IFN-γ ELISpot analysis using 6 peptide pools covering the Spike protein. Values represented the average response in each group one week after the third immunization. Standard error is shown in error bar. (C and D) Properties of MERS-Spike specific dominant epitope. Secretion of IFN-γ by splenocytes from MERS-Spike specific immune responses using matrix pooling of peptides. These results were expressed as the number of IFN-γ secreting cells / 10 ⁶ splenocytes after subtracting the value without peptide stimulation (mean ± SD of 3 mice per group). Similar values were obtained from two separate experiments. Figure 2 shows the functional profile of the cellular immune response elicited by MERS-HCoV vaccine. (A) As shown, C57 / BL6 mice group (n = 9 / group) were immunized 3 times with 2 weeks of Spike-Wt and SpikeΔCD DNA vaccine, respectively. Samples were collected one week after the third immunization. (B) Spike-specific T lymphocyte responses were assessed using IFN-γ ELISpot analysis using 6 peptide pools covering the Spike protein. Values represented the average response in each group one week after the third immunization. Standard error is shown in error bar. (C and D) Properties of MERS-Spike specific dominant epitope. Secretion of IFN-γ by splenocytes from MERS-Spike specific immune responses using matrix pooling of peptides. These results were expressed as the number of IFN-γ secreting cells / 10 ⁶ splenocytes after subtracting the value without peptide stimulation (mean ± SD of 3 mice per group). Similar values were obtained from two separate experiments. Figure 2 shows the functional profile of the cellular immune response elicited by MERS-HCoV vaccine. (A) As shown, C57 / BL6 mice group (n = 9 / group) were immunized 3 times with 2 weeks of Spike-Wt and SpikeΔCD DNA vaccine, respectively. Samples were collected one week after the third immunization. (B) Spike-specific T lymphocyte responses were assessed using IFN-γ ELISpot analysis using 6 peptide pools covering the Spike protein. Values represented the average response in each group one week after the third immunization. Standard error is shown in error bar. (C and D) Properties of MERS-Spike specific dominant epitope. Secretion of IFN-γ by splenocytes from MERS-Spike specific immune responses using matrix pooling of peptides. These results were expressed as the number of IFN-γ secreting cells / 10 ⁶ splenocytes after subtracting the value without peptide stimulation (mean ± SD of 3 mice per group). Similar values were obtained from two separate experiments. Shows systemic anti-MERS-HCoV IgG levels after DNA immunization. (A) Mouse serum anti-IgG response measured by ELISA at the indicated serum dilutions against MERS-Spike as the coating antigen. Pooled sera from individual groups of mice (one week after the third immunization) were serially diluted and MERS-Spike specific total IgG was measured by ELISA. Each curve represented the OD value of each group of 9 mice given Spike-Wt DNA vaccine, SpikeΔCD-Spike DNA vaccine, or empty DNA vector, as indicated. Each data point was the mean absorbance from 9 mice. (B) The endpoint titer against the serum of mice given Spike-Wt immunity was calculated at the indicated time points. (C) Western blot analysis of immunized serum binding to recombinant Spike protein at the indicated concentration (μg). Sera pooled from Spike-Wt immunized mice was used as a primary antibody at a 1: 100 dilution. (D) Neutralizing antibody reaction detected by virus infection analysis. Sera one week after the third immunization were collected from the immunized mice. Neutralizing antibody titers were measured at 50% inhibition (IC ₅₀ ) of virus infection on target cells. The data shown was the geometric mean titer and standard deviation for each group. Statistical differences between each test group were measured and p-values were indicated. (E) Neutralization of MERS-Spike pseudotyped virus infectivity by antisera obtained from mice after receiving three injections of Spike-Wt DNA or pVax1 DNA. IC ₅₀ was defined as the reciprocal of the antiserum dilution at which virus entry was inhibited by 50% (dashed line). Data from 4 mice per group are shown. Shows systemic anti-MERS-HCoV IgG levels after DNA immunization. (A) Mouse serum anti-IgG response measured by ELISA at the indicated serum dilutions against MERS-Spike as the coating antigen. Pooled sera from individual groups of mice (one week after the third immunization) were serially diluted and MERS-Spike specific total IgG was measured by ELISA. Each curve represented the OD value of each group of 9 mice given Spike-Wt DNA vaccine, SpikeΔCD-Spike DNA vaccine, or empty DNA vector, as indicated. Each data point was the mean absorbance from 9 mice. (B) The endpoint titer against the serum of mice given Spike-Wt immunity was calculated at the indicated time points. (C) Western blot analysis of immunized serum binding to recombinant Spike protein at the indicated concentration (μg). Sera pooled from Spike-Wt immunized mice was used as a primary antibody at a 1: 100 dilution. (D) Neutralizing antibody reaction detected by virus infection analysis. Sera one week after the third immunization were collected from the immunized mice. Neutralizing antibody titers were measured at 50% inhibition (IC ₅₀ ) of virus infection on target cells. The data shown was the geometric mean titer and standard deviation for each group. Statistical differences between each test group were measured and p-values were indicated. (E) Neutralization of MERS-Spike pseudotyped virus infectivity by antisera obtained from mice after receiving three injections of Spike-Wt DNA or pVax1 DNA. IC ₅₀ was defined as the reciprocal of the antiserum dilution at which virus entry was inhibited by 50% (dashed line). Data from 4 mice per group are shown. Shows systemic anti-MERS-HCoV IgG levels after DNA immunization. (A) Mouse serum anti-IgG response measured by ELISA at the indicated serum dilutions against MERS-Spike as the coating antigen. Pooled sera from individual groups of mice (one week after the third immunization) were serially diluted and MERS-Spike specific total IgG was measured by ELISA. Each curve represented the OD value of each group of 9 mice given Spike-Wt DNA vaccine, SpikeΔCD-Spike DNA vaccine, or empty DNA vector, as indicated. Each data point was the mean absorbance from 9 mice. (B) The endpoint titer against the serum of mice given Spike-Wt immunity was calculated at the indicated time points. (C) Western blot analysis of immunized serum binding to recombinant Spike protein at the indicated concentration (μg). Sera pooled from Spike-Wt immunized mice was used as a primary antibody at a 1: 100 dilution. (D) Neutralizing antibody reaction detected by virus infection analysis. Sera one week after the third immunization were collected from the immunized mice. Neutralizing antibody titers were measured at 50% inhibition (IC ₅₀ ) of virus infection on target cells. The data shown was the geometric mean titer and standard deviation for each group. Statistical differences between each test group were measured and p-values were indicated. (E) Neutralization of MERS-Spike pseudotyped virus infectivity by antisera obtained from mice after receiving three injections of Spike-Wt DNA or pVax1 DNA. IC ₅₀ was defined as the reciprocal of the antiserum dilution at which virus entry was inhibited by 50% (dashed line). Data from 4 mice per group are shown. Shows systemic anti-MERS-HCoV IgG levels after DNA immunization. (A) Mouse serum anti-IgG response measured by ELISA at the indicated serum dilutions against MERS-Spike as the coating antigen. Pooled sera from individual groups of mice (one week after the third immunization) were serially diluted and MERS-Spike specific total IgG was measured by ELISA. Each curve represented the OD value of each group of 9 mice given Spike-Wt DNA vaccine, SpikeΔCD-Spike DNA vaccine, or empty DNA vector, as indicated. Each data point was the mean absorbance from 9 mice. (B) The endpoint titer against the serum of mice given Spike-Wt immunity was calculated at the indicated time points. (C) Western blot analysis of immunized serum binding to recombinant Spike protein at the indicated concentration (μg). Sera pooled from Spike-Wt immunized mice was used as a primary antibody at a 1: 100 dilution. (D) Neutralizing antibody reaction detected by virus infection analysis. Sera one week after the third immunization were collected from the immunized mice. Neutralizing antibody titers were measured at 50% inhibition (IC ₅₀ ) of virus infection on target cells. The data shown was the geometric mean titer and standard deviation for each group. Statistical differences between each test group were measured and p-values were indicated. (E) Neutralization of MERS-Spike pseudotyped virus infectivity by antisera obtained from mice after receiving three injections of Spike-Wt DNA or pVax1 DNA. IC ₅₀ was defined as the reciprocal of the antiserum dilution at which virus entry was inhibited by 50% (dashed line). Data from 4 mice per group are shown. Shows systemic anti-MERS-HCoV IgG levels after DNA immunization. (A) Mouse serum anti-IgG response measured by ELISA at the indicated serum dilutions against MERS-Spike as the coating antigen. Pooled sera from individual groups of mice (one week after the third immunization) were serially diluted and MERS-Spike specific total IgG was measured by ELISA. Each curve represented the OD value of each group of 9 mice given Spike-Wt DNA vaccine, SpikeΔCD-Spike DNA vaccine, or empty DNA vector, as indicated. Each data point was the mean absorbance from 9 mice. (B) The endpoint titer against the serum of mice given Spike-Wt immunity was calculated at the indicated time points. (C) Western blot analysis of immunized serum binding to recombinant Spike protein at the indicated concentration (μg). Sera pooled from Spike-Wt immunized mice was used as a primary antibody at a 1: 100 dilution. (D) Neutralizing antibody reaction detected by virus infection analysis. Sera one week after the third immunization were collected from the immunized mice. Neutralizing antibody titers were measured at 50% inhibition (IC ₅₀ ) of virus infection on target cells. The data shown was the geometric mean titer and standard deviation for each group. Statistical differences between each test group were measured and p-values were indicated. (E) Neutralization of MERS-Spike pseudotyped virus infectivity by antisera obtained from mice after receiving three injections of Spike-Wt DNA or pVax1 DNA. IC ₅₀ was defined as the reciprocal of the antiserum dilution at which virus entry was inhibited by 50% (dashed line). Data from 4 mice per group are shown. Figure 3 shows the analysis of interferon-gamma (IFN-γ) producing cells induced by MERS-Spike DNA vaccine in non-human primates (NHP). (A) Study design, vaccine and challenge regimen in rhesus monkeys. Three groups (low, high and control) rhesus monkeys (n = 4 / each group) were immunized with MERS-Spike DNA at 0, 3, and 6 weeks (wks) as indicated. IFN-γ ELISpot, intracellular cytokine staining and antibody binding, and Nab analysis were performed from the third immunized specimen. A biopsy was performed after the MERS challenge. (B) ELISPOT analysis of cells from monkeys immunized with MERS-Spike DNA. PBMC were isolated from each of the monkeys immunized with DNA and used for ELISPOT analysis to detect IFN-γ producing cells responding to the MERS-Spike peptide pool for 24 hours as described in Example 9. The frequency of MERS-Spike specific IFN-γ secreting cells / 10 ⁶ PBMC was determined by ELISpot analysis. Results are expressed as mean ± SEM. (C) Frequency of vaccine-derived cytokine (IFN-γ, IL-2, or TNF-α) producing CD4 ⁺ and CD8 ⁺ T cells. Monkey PBMC's (n = 4) were isolated after the last DNA immunization and stimulated in vitro with pooled MERS-Spike peptides. Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2 and then analyzed by multiparameter flow cytometry. Figure 3 shows the analysis of interferon-gamma (IFN-γ) producing cells induced by MERS-Spike DNA vaccine in non-human primates (NHP). (A) Study design, vaccine and challenge regimen in rhesus monkeys. Three groups (low, high and control) rhesus monkeys (n = 4 / each group) were immunized with MERS-Spike DNA at 0, 3, and 6 weeks (wks) as indicated. IFN-γ ELISpot, intracellular cytokine staining and antibody binding, and Nab analysis were performed from the third immunized specimen. A biopsy was performed after the MERS challenge. (B) ELISPOT analysis of cells from monkeys immunized with MERS-Spike DNA. PBMC were isolated from each of the monkeys immunized with DNA and used for ELISPOT analysis to detect IFN-γ producing cells responding to the MERS-Spike peptide pool for 24 hours as described in Example 9. The frequency of MERS-Spike specific IFN-γ secreting cells / 10 ⁶ PBMC was determined by ELISpot analysis. Results are expressed as mean ± SEM. (C) Frequency of vaccine-derived cytokine (IFN-γ, IL-2, or TNF-α) producing CD4 ⁺ and CD8 ⁺ T cells. Monkey PBMC's (n = 4) were isolated after the last DNA immunization and stimulated in vitro with pooled MERS-Spike peptides. Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2 and then analyzed by multiparameter flow cytometry. Figure 3 shows the analysis of interferon-gamma (IFN-γ) producing cells induced by MERS-Spike DNA vaccine in non-human primates (NHP). (A) Study design, vaccine and challenge regimen in rhesus monkeys. Three groups (low, high and control) rhesus monkeys (n = 4 / each group) were immunized with MERS-Spike DNA at 0, 3, and 6 weeks (wks) as indicated. IFN-γ ELISpot, intracellular cytokine staining and antibody binding, and Nab analysis were performed from the third immunized specimen. A biopsy was performed after the MERS challenge. (B) ELISPOT analysis of cells from monkeys immunized with MERS-Spike DNA. PBMC were isolated from each of the monkeys immunized with DNA and used for ELISPOT analysis to detect IFN-γ producing cells responding to the MERS-Spike peptide pool for 24 hours as described in Example 9. The frequency of MERS-Spike specific IFN-γ secreting cells / 10 ⁶ PBMC was determined by ELISpot analysis. Results are expressed as mean ± SEM. (C) Frequency of vaccine-derived cytokine (IFN-γ, IL-2, or TNF-α) producing CD4 ⁺ and CD8 ⁺ T cells. Monkey PBMC's (n = 4) were isolated after the last DNA immunization and stimulated in vitro with pooled MERS-Spike peptides. Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2 and then analyzed by multiparameter flow cytometry. Shown are antibody binding titers and neutralizing antibody (Nab) response after MERS-Spike DNA vaccination. (A and B) MERS specific antibodies in serum binding and IgG specific endpoint titers at various times after immunization. Serum was collected after each vaccination and endpoint titer was calculated by ELISA. (C and D) Effect of DNA vaccination and characteristics of neutralizing antibody response. Serum specimens were collected from animals before each immunization and 2 weeks after the last vaccination. These sera were analyzed for neutralizing antibodies. Neutralization with live virus (C) or MERS pseudovirus (D). Serially diluted immunized sera were tested with MERS virus. For pseudovirus neutralization analysis, anti-CHIKV monkey serum was used as a negative antibody control and VSV-G pseudotyped virus was used as a pseudovirus control for neutralization specificity. Specimens were tested by PRNT50 analysis with MERS virus. Shown are antibody binding titers and neutralizing antibody (Nab) response after MERS-Spike DNA vaccination. (A and B) MERS specific antibodies in serum binding and IgG specific endpoint titers at various times after immunization. Serum was collected after each vaccination and endpoint titer was calculated by ELISA. (C and D) Effect of DNA vaccination and characteristics of neutralizing antibody response. Serum specimens were collected from animals before each immunization and 2 weeks after the last vaccination. These sera were analyzed for neutralizing antibodies. Neutralization with live virus (C) or MERS pseudovirus (D). Serially diluted immunized sera were tested with MERS virus. For pseudovirus neutralization analysis, anti-CHIKV monkey serum was used as a negative antibody control and VSV-G pseudotyped virus was used as a pseudovirus control for neutralization specificity. Specimens were tested by PRNT50 analysis with MERS virus. Shown are antibody binding titers and neutralizing antibody (Nab) response after MERS-Spike DNA vaccination. (A and B) MERS specific antibodies in serum binding and IgG specific endpoint titers at various times after immunization. Serum was collected after each vaccination and endpoint titer was calculated by ELISA. (C and D) Effect of DNA vaccination and characteristics of neutralizing antibody response. Serum specimens were collected from animals before each immunization and 2 weeks after the last vaccination. These sera were analyzed for neutralizing antibodies. Neutralization with live virus (C) or MERS pseudovirus (D). Serially diluted immunized sera were tested with MERS virus. For pseudovirus neutralization analysis, anti-CHIKV monkey serum was used as a negative antibody control and VSV-G pseudotyped virus was used as a pseudovirus control for neutralization specificity. Specimens were tested by PRNT50 analysis with MERS virus. Shown are antibody binding titers and neutralizing antibody (Nab) response after MERS-Spike DNA vaccination. (A and B) MERS specific antibodies in serum binding and IgG specific endpoint titers at various times after immunization. Serum was collected after each vaccination and endpoint titer was calculated by ELISA. (C and D) Effect of DNA vaccination and characteristics of neutralizing antibody response. Serum specimens were collected from animals before each immunization and 2 weeks after the last vaccination. These sera were analyzed for neutralizing antibodies. Neutralization with live virus (C) or MERS pseudovirus (D). Serially diluted immunized sera were tested with MERS virus. For pseudovirus neutralization analysis, anti-CHIKV monkey serum was used as a negative antibody control and VSV-G pseudotyped virus was used as a pseudovirus control for neutralization specificity. Specimens were tested by PRNT50 analysis with MERS virus. FIG. 25 shows the functional profile of CD4 ⁺ and CD8 ⁺ T cell responses elicited by the MERS-Spike vaccine. The cytokine profiles of specific CD4 ⁺ T cells and CD8 ⁺ T cells induced after vaccination are shown in (A) and (B), respectively. Mouse splenocytes (n = 3) were isolated one week after the last DNA vaccine immunization and stimulated in vitro with pooled MERS-Spike peptides. Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2 and then analyzed by FACS. (A and B) Scatter plot showing MERS-specific CD4 ⁺ T and CD8 ⁺ T cells releasing IFN-γ, TNF-α, IL-2, and dual IFN-γ / TNF-α cytokines. (C and D) Bar graphs show multifunctional subpopulations of single, double, and triple positive CD4 ⁺ and CD8 ⁺ T cells that release the cytokines IFN-γ, TNF-α, and IL-2. The pie chart shows the percentage of each cytokine subpopulation. Data were expressed as mean ± SEM of 3 mice per group. FIG. 25 shows the functional profile of CD4 ⁺ and CD8 ⁺ T cell responses elicited by the MERS-Spike vaccine. The cytokine profiles of specific CD4 ⁺ T cells and CD8 ⁺ T cells induced after vaccination are shown in (A) and (B), respectively. Mouse splenocytes (n = 3) were isolated one week after the last DNA vaccine immunization and stimulated in vitro with pooled MERS-Spike peptides. Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2 and then analyzed by FACS. (A and B) Scatter plot showing MERS-specific CD4 ⁺ T and CD8 ⁺ T cells releasing IFN-γ, TNF-α, IL-2, and dual IFN-γ / TNF-α cytokines. (C and D) Bar graphs show multifunctional subpopulations of single, double, and triple positive CD4 ⁺ and CD8 ⁺ T cells that release the cytokines IFN-γ, TNF-α, and IL-2. The pie chart shows the percentage of each cytokine subpopulation. Data were expressed as mean ± SEM of 3 mice per group. FIG. 25 shows the functional profile of CD4 ⁺ and CD8 ⁺ T cell responses elicited by the MERS-Spike vaccine. The cytokine profiles of specific CD4 ⁺ T cells and CD8 ⁺ T cells induced after vaccination are shown in (A) and (B), respectively. Mouse splenocytes (n = 3) were isolated one week after the last DNA vaccine immunization and stimulated in vitro with pooled MERS-Spike peptides. Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2 and then analyzed by FACS. (A and B) Scatter plot showing MERS-specific CD4 ⁺ T and CD8 ⁺ T cells releasing IFN-γ, TNF-α, IL-2, and dual IFN-γ / TNF-α cytokines. (C and D) Bar graphs show multifunctional subpopulations of single, double, and triple positive CD4 ⁺ and CD8 ⁺ T cells that release the cytokines IFN-γ, TNF-α, and IL-2. The pie chart shows the percentage of each cytokine subpopulation. Data were expressed as mean ± SEM of 3 mice per group. FIG. 25 shows the functional profile of CD4 ⁺ and CD8 ⁺ T cell responses elicited by the MERS-Spike vaccine. The cytokine profiles of specific CD4 ⁺ T cells and CD8 ⁺ T cells induced after vaccination are shown in (A) and (B), respectively. Mouse splenocytes (n = 3) were isolated one week after the last DNA vaccine immunization and stimulated in vitro with pooled MERS-Spike peptides. Cells were stained for intracellular production of IFN-γ, TNF-α, and IL-2 and then analyzed by FACS. (A and B) Scatter plot showing MERS-specific CD4 ⁺ T and CD8 ⁺ T cells releasing IFN-γ, TNF-α, IL-2, and dual IFN-γ / TNF-α cytokines. (C and D) Bar graphs show multifunctional subpopulations of single, double, and triple positive CD4 ⁺ and CD8 ⁺ T cells that release the cytokines IFN-γ, TNF-α, and IL-2. The pie chart shows the percentage of each cytokine subpopulation. Data were expressed as mean ± SEM of 3 mice per group. FIG. 6 shows inhibition of apoptosis induced by MERS-S pseudovirus by mouse immune serum immunized with Spike DNA. (A) MERS-HCoV-Spike mouse serum neutralizes MERS in vitro and inhibits syncytium formation. Uninfected Vero cells represented negative control, and positive control represented those cells infected with MERS-HCoV pseudovirus. Syncytium formation was indicated by an arrow. Syncytium formation was observed under the microscope 36 hours after infection. (B) MERS-Spike pseudovirus pre-cultured in the presence of either 1: 100 dilution of pVax1 or MERS-Spike immunized serum and added to Vero cells. Two days after infection, the rate of cell death was measured by Annexin V / PI staining. Bar graphs showed MERS pseudovirus infection values from FACS data from a single experiment performed in triplicate. Similar results were obtained from three independent experiments. FIG. 6 shows inhibition of apoptosis induced by MERS-S pseudovirus by mouse immune serum immunized with Spike DNA. (A) MERS-HCoV-Spike mouse serum neutralizes MERS in vitro and inhibits syncytium formation. Uninfected Vero cells represented negative control, and positive control represented those cells infected with MERS-HCoV pseudovirus. Syncytium formation was indicated by an arrow. Syncytium formation was observed under the microscope 36 hours after infection. (B) MERS-Spike pseudovirus pre-cultured in the presence of either 1: 100 dilution of pVax1 or MERS-Spike immunized serum and added to Vero cells. Two days after infection, the rate of cell death was measured by Annexin V / PI staining. Bar graphs showed MERS pseudovirus infection values from FACS data from a single experiment performed in triplicate. Similar results were obtained from three independent experiments. Shows antibody response after MERS-Spike DNA vaccination in combination with electroporation. Sera from monkeys vaccinated with pVax1, pMERS-Spike (0.5 mg / animal; low dose), or pMERS-Spike (2 mg / animal; high dose) were collected 2 weeks after the third immunization. These sera were analyzed for antibodies conjugated to MERS-Spike protein. These analysis results are shown in (A) and (B). Shows antibody response after MERS-Spike DNA vaccination in combination with electroporation. Sera from monkeys vaccinated with pVax1, pMERS-Spike (0.5 mg / animal; low dose), or pMERS-Spike (2 mg / animal; high dose) were collected 2 weeks after the third immunization. These sera were analyzed for antibodies conjugated to MERS-Spike protein. These analysis results are shown in (A) and (B). The results of MERS challenge after vaccination and the pathobiology are shown. (A) Average viral load measured by qRT-PCR from individual tissues collected at necropsy. (B) Average viral load of all germ layers bound is shown in the inset. Log TCID 50 eq / g, log TCID 50 equivalents per gram of tissue; from 3 animals per group, 1 specimen per tissue per animal was analyzed. The results of MERS challenge after vaccination and the pathobiology are shown. (A) Average viral load measured by qRT-PCR from individual tissues collected at necropsy. (B) Average viral load of all germ layers bound is shown in the inset. Log TCID 50 eq / g, log TCID 50 equivalents per gram of tissue; from 3 animals per group, 1 specimen per tissue per animal was analyzed.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a vaccine comprising a Middle Eastern respiratory syndrome coronavirus (MERS-CoV) antigen. MERS-CoV is a new type of highly pathogenic virus that first appeared in 2012, and therefore the vaccine described herein is one of the first vaccines against the target MERS-CoV. Thus, the vaccine provides a treatment for this new pathogenic virus where there is no previous treatment and the possibility of a pandemic remains.

  The MERS-CoV antigen can be a MERS-CoV consensus spike antigen. The MERS-CoV consensus spike antigen can be derived from the sequence of the spike antigen of the MERS-CoV strain, and thus the MERS-CoV consensus spike antigen is non-repetitive. The MERS-CoV consensus spike antigen may be deficient in the cytoplasmic domain. Therefore, the vaccine of the present invention can be widely applied to various strains of MERS-CoV because of the non-repetitive sequence of the MERS-CoV consensus spike antigen. These non-repetitive sequences make the vaccine universally protective against various strains of MERS-CoV, including genetically different variants of MERS-CoV.

The vaccine can be used to protect against MERS-CoV and to handle any strain of MERS-CoV. The vaccine can elicit both humoral and cellular immune responses that target the MERS-CoV spike antigen. The vaccine can elicit neutralizing antibodies and immunoglobulin G (IgG) antibodies reactive with MERS-CoV spike antigen. The vaccine is reactive with MERS-CoV spike antigen and produces CD8 ⁺ and CD4 producing interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) ^{+ A} T cell response can also be elicited.

1. Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, but methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned in this document are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not limiting.

  In this document, “compose”, “comprise”, “comprise”, “have”, “can”, “include”, and variants of these are unrestricted transition phrases, terms, or words. It does not exclude the possibility of further actions or structures. The singular forms “indefinite” and “definite” include plural references unless the context clearly indicates otherwise. This disclosure also contemplates the embodiments presented herein or other elements “comprising”, “consisting of”, and “consisting essentially of” with or without the explicit description.

  As used herein, “adjuvant” refers to any molecule that is added to the vaccines described herein to enhance the immunogenicity of the antigen.

As used herein, “antibody” refers to class IgG, IgM, IgA, IgD, or IgE, or fragments, Fab, F (ab ′) ₂ , Fd, and fragments or derivatives thereof including single chain antibodies, diabodies, It refers to bispecific antibodies, bivalent antibodies, and derivatives thereof. The antibody is an antibody isolated from a mammalian serum sample, a polyclonal antibody, an affinity purified antibody, or a mixture thereof, which exhibits sufficient binding specificity for a desired epitope or a sequence derived therefrom. Good.

  As used herein, “coding sequence” or “encoding nucleic acid” refers to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence encoding a protein. The coding sequence may further include initiation and termination signals operably linked to regulatory elements including a promoter capable of directing expression in an individual or mammalian cell to which the nucleic acid has been administered and a polyadenylation signal.

  As used herein, “complement” or “complementary” refers to Watson-Crick (eg, AT / U and CG) or Hoogsteen-type base pairs between nucleotides or nucleotide analogs of a nucleic acid molecule.

  As used herein, “consensus” or “consensus sequence” can mean a synthetic nucleic acid sequence constructed based on an alignment analysis of multiple subtypes of a particular antigen, or a corresponding polypeptide sequence. The sequence can be used to induce broad immunity against multiple subtypes, serotypes, or strains of a particular antigen. Synthetic antigens such as fusion proteins can be engineered to generate consensus sequences (or consensus antigens).

  As used interchangeably herein, “electroporation”, “electropermeabilization”, or “electrokinetic enhancement (“ EP ”)” is used to induce microscopic passageways (pores) in biological membranes. It refers to the use of transmembrane electric field pulses. Their presence allows the passage of biomolecules such as plasmids, oligonucleotides, siRNA, drugs, ions, and water from one side of the cell membrane to the other.

  As used herein, “fragment” refers to a nucleic acid sequence or portion thereof that encodes a polypeptide capable of eliciting an immune response in a mammal. The fragment may be a DNA fragment selected from at least one of various nucleotide sequences encoding the protein fragments described below.

  A “fragment” or “immunogenic fragment” involving a polypeptide sequence refers to a polypeptide capable of eliciting an immune response in a mammal that cross-reacts with a fully wild-type MERS-CoV antigen. A fragment of a consensus protein is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or more of the consensus protein, or at least 95 % May constitute. In some embodiments, the consensus protein fragment is at least 20 amino acids or more, at least 30 amino acids or more, at least 40 amino acids or more, at least 50 amino acids or more, at least 60 of the consensus protein. Amino acids or more, at least 70 amino acids or more, at least 80 amino acids or more, at least 90 amino acids or more, at least 100 amino acids or more, at least 110 amino acids or more, at least 120 amino acids Or more, at least 130 amino acids or more, at least 140 amino acids or more, at least 150 amino acids Is at least 160 amino acids or more, at least 170 amino acids or more, at least 180 amino acids or more, at least 190 amino acids or more, at least 200 amino acids or more, at least 210 amino acids or more Thus, it may comprise at least 220 amino acids or more, at least 230 amino acids or more, or at least 240 amino acids or more.

  As used herein, the term “genetic construct” refers to a DNA or RNA molecule comprising a nucleotide sequence encoding a protein. The coding sequence includes initiation and termination signals operably linked to regulatory elements including a promoter capable of directing expression in a cell of the individual to which the nucleic acid molecule has been administered and a polyadenylation signal. As used herein, the term “expressible form” refers to a gene that includes an essential regulatory element operably linked to a coding sequence that encodes a protein so that the coding sequence is expressed when present in the cells of the individual. Refers to a construct.

  As used herein, “homology” or “homology” in two or more nucleic acid or polypeptide sequences means that the sequences have a certain percentage of residues that are identical over a particular region. The ratio is determined by aligning the two sequences optimally, comparing the two sequences over the specific region, measuring the number of positions where homologous residues occur in both sequences, and determining the number of matching positions. Is divided by the total number of positions in the specific region, and the result is multiplied by 100 to obtain the percentage of sequence homology. If the two sequences are of different lengths, or if the alignment produces one or more sticky ends and the comparison specific region contains only a single sequence, then the single sequence residues are not computational molecules Include in denominator. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Homology may be performed manually or using a computer sequence algorithm such as BLAST or BLAST 2.0.

  As used herein, “immune response” refers to the activation of the immune system of a host, eg, a mammal, in response to introduction of an antigen. The immune response can be in the form of a cellular or humoral response, or both.

  As used herein, “nucleic acid” or “oligonucleotide” or “polynucleotide” refers to at least two nucleotides covalently linked together. Single-strand drawing also determines the complementary strand sequence. Thus, the nucleic acid also covers the depicted single-stranded complementary strand. Many variants of a nucleic acid can be used for the same purpose as a given nucleic acid. Thus, a nucleic acid also encompasses a substantially homologous nucleic acid and its complement. The single strand provides a probe that can hybridize to the target sequence under stringent hybridization conditions. Thus, nucleic acids also cover probes that hybridize under severe hybridization conditions.

  Nucleic acids may be single stranded or double stranded and may contain both double stranded and single stranded sequence portions. The nucleic acid can be DNA, both genomic DNA and cDNA, RNA, or a hybrid, and the nucleic acid can be a combination of deoxyribonucleotides and ribonucleotides, and uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, A combination of bases including hypoxanthine, isocytosine, and isoguanine may be included. Nucleic acids can be obtained by chemical synthesis or recombinant methods.

  As used herein, “operably linked” means that gene expression is governed by a spatially connected promoter. A promoter may be located under its control 5 '(upstream) or 3' (downstream) of a gene. The distance between the promoter and the gene may be approximately the same as the distance between the promoter that controls the gene from which the promoter is derived. As is well known in the art, this variation in distance can be adjusted without loss of promoter function.

  As used herein, “peptide”, “protein”, or “polypeptide” can refer to a binding sequence of amino acids and can be natural, synthetic, modified, or a combination of natural and synthetic.

  As used herein, “promoter” refers to a synthetic or naturally occurring molecule that allows conferring, activating, or enhancing expression of a nucleic acid in a cell. A promoter may include one or more specific transcription control sequences to further enhance expression and / or modify its spatial and / or temporal expression. A promoter may include distal enhancer or repressor elements that may be thousands of base pairs away from the transcription start site. The promoter may be derived from sources including viruses, bacteria, molds, plants, insects, and animals. Promoters are for cells, tissues, or organs where expression occurs, or for developmental stages where expression occurs, or in response to external stimuli such as physiological stress, pathogens, metal ions, or inducers, The structural or specific expression of genetic elements can be regulated. Representative examples of promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV40 The late promoter and the CMV IE promoter can be mentioned.

  “Signal peptide” and “leader sequence” are used interchangeably herein and refer to an amino acid sequence capable of binding at the amino terminus of a MERS-CoV protein described herein. The signal peptide / leader sequence usually directs protein localization. The signal peptide / leader sequence herein preferably facilitates secretion of the protein from the cell in which the protein is produced. The signal peptide / leader sequence is frequently cleaved from the rest of the protein, often referred to as the mature protein, upon secretion from the cell. A signal peptide / leader sequence binds at the N-terminus of the protein.

  As used herein, “patient” can refer to a mammal in need of or immunized with the vaccines described herein. The mammal can be a human, chimpanzee, dog, cat, horse, cow, mouse, or rat.

  In this document, “substantially homologous” means that the first and second amino acid sequences are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or more amino acid regions, at least 60%, 65%, 70%, 75%, 80%, 81%, 82 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, Or it can mean 99%. Substantially homologous means that the first nucleic acid sequence and the second nucleic acid sequence are 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, Over at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83 over a region of 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or more nucleotides %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% It can also mean that.

  As used herein, “treatment” or “treating” can mean protecting an animal from the disease by preventing, suppressing, suppressing, or completely eliminating the disease. Prevention of the disease involves administering the vaccine of the present invention to the animal before the disease develops. Disease suppression includes administration of the vaccine of the present invention to an animal after the induction of the disease but before clinical findings appear. Disease suppression involves administering the vaccine of the present invention to an animal after clinical manifestations of the disease have emerged.

  As used herein, a “variant” relating to a nucleic acid refers to (i) a portion or fragment of a referenced nucleotide sequence; (ii) a referenced nucleotide sequence or the complement of a portion thereof; (iii) a referenced nucleic acid or A nucleic acid that is substantially homologous to its complement; or (iv) a nucleic acid that hybridizes to a referenced nucleic acid under stringent conditions, its complement, or a sequence that is substantially homologous to them.

  A variant can be further defined as a peptide or polypeptide that differs in amino acid sequence by amino acid insertion, deletion, or conservative substitution but retains at least one biological activity. Representative examples of “biological activity” include the ability to bind by specific antibodies or the ability to promote immune responses. A variant can also refer to a protein having an amino acid sequence substantially homologous to a referenced protein having an amino acid sequence that retains at least one biological activity. A conservative substitution of amino acids, ie, substitution of an amino acid with another amino acid having similar properties (eg, hydrophilicity, charged region extent and distribution) is generally recognized in the art as accompanied by minor changes. Is done. These minor changes are, of course, in the art, but can be identified in part by considering the hydrophobic hydrophilicity index of the amino acid. Kyte et al. , J .; Mol. Biol. 157: 105-132 (1982). The hydrophobic hydrophilicity index of an amino acid is based on a consideration of its hydrophobicity and changes. It is well known in the art that amino acids of similar hydrophobic hydrophilicity indices can be substituted and protein function can still be maintained. In one embodiment, amino acids with a hydrophobic hydrophilicity index of ± 2 are substituted. The hydrophilicity of amino acids can also be used to represent substitutions that will result in proteins that retain biological function. The consideration of the hydrophilicity of the amino acids in the peptide allows the calculation of the maximum local average hydrophilicity of the peptide, a useful measure that has been reported to be associated with antigenicity and immunogenicity. Substitution of amino acids with similar hydrophilicity values can, of course, result in peptides that retain biological activity, eg, immunogenicity, as is well known in the art. Substitutions can be made between amino acids whose hydrophilicity values are within ± 2 of each other. Both the hydrophobicity index and the hydrophilicity value of an amino acid are influenced by the specific side chain of that amino acid. According to that observation, amino acid substitutions corresponding to biological functions are related to the relative similarity of these amino acids, and in particular to the side of these amino acids, as represented by hydrophobicity, hydrophilicity, charge, size, and other properties. It is understood according to the chain.

  A variant can be a nucleic acid sequence that is substantially homologous over the entire length of the entire gene sequence or a fragment thereof. The nucleic acid sequence is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% over the entire length of the gene sequence or fragment thereof. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous. A variant can be an amino acid sequence that is substantially homologous over the entire length of the amino acid sequence or fragment thereof. The amino acid sequence is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% over the entire length of the amino acid sequence or fragment thereof. 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous.

  As used herein, “vector” refers to a nucleic acid sequence that includes an origin of replication. The vector may be a viral vector, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. The vector may be a DNA or RNA vector. The vector may be a self-replicating extrachromosomal vector, preferably a DNA plasmid.

  For the enumeration of numerical ranges in this document, each intervening numerical value between them with the same accuracy is explicitly considered. For example, for the range 6-9, the values 7 and 8 are considered in addition to 6 and 9, and for the range 6.0-7.0, 6.0, 6.1, 6.2, 6 .3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly considered.

2. Vaccines In this document, immunogenic compositions such as vaccines comprising a Middle East respiratory syndrome coronavirus (MERS-CoV) antigen, fragments thereof, variants thereof, or combinations thereof are provided. The vaccine can be used to protect against any strain of MERS-CoV, thereby treating, preventing, and / or protecting from MERS-CoV based lesions. The vaccine significantly induces an immune response in patients receiving the vaccine and prevents and treats MERS-CoV infection.

  The vaccine may be a DNA vaccine, a peptide vaccine, or a mixed DNA and peptide vaccine. The DNA vaccine can comprise a nucleic acid sequence encoding a MERS-CoV antigen. The nucleic acid sequence can be DNA, RNA, cDNA, variants thereof, fragments thereof, or combinations thereof. The nucleic acid sequence can also include additional sequences encoding linker, leader, or tag sequences linked to the MERS-CoV antigen by peptide bonds. The peptide vaccine may comprise a MERS-CoV antigenic peptide, MERS-CoV antigenic protein, variants thereof, fragments thereof, or combinations thereof. The mixed DNA and peptide vaccine may comprise the above-described nucleic acid sequence encoding the MERS-CoV antigen and the MERS-CoV antigenic peptide or protein, and the MERS-CoV antigenic peptide or protein and the encoded MERS- CoV antigens have the same amino acid sequence.

  The vaccine can induce a humoral immune response in patients receiving the vaccine. The induced humoral immune response may be specific for the MERS-CoV antigen. The induced humoral immune response can be reactive with the MERS-CoV antigen. The humoral immune response can be induced in patients who have been administered the vaccine from about 1.5 times to about 16 times, from about 2 times to about 12 times, or from about 3 times to about 10 times. The humoral immune response comprises at least about 1.5 fold, at least about 2.0 fold, at least about 2.5 fold, at least about 3.0 fold, at least about 3.5 fold, at least about 4. 0 times, at least about 4.5 times, at least about 5.0 times, at least about 5.5 times, at least about 6.0 times, at least about 6.5 times, at least about 7.0 times, at least about 7.5 times Times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, at least about 9.5 times, at least about 10.0 times, at least about 10.5 times, at least about 11.0 times At least about 11.5 times, at least about 12.0 times, at least about 12.5 times, at least about 13.0 times, at least about 13.5 times, at least about 14.0 times, at least about 14.5 times, At least about 15.0 It can be induced at least about 15.5 fold, or were administered at least about 16.0 times the patient.

The humoral immune response induced with the vaccine may include an increase in neutralizing antibody concentration in patients receiving the vaccine compared to patients not receiving the vaccine. The neutralizing antibody can be specific for the MERS-CoV antigen. The neutralizing antibody can be reactive with the MERS-CoV antigen. The neutralizing antibody may provide prevention and / or treatment of MERS-CoV infection and related lesions in patients receiving the vaccine.

  The humoral immune response induced with the vaccine may include an increase in IgG antibody concentration in patients receiving the vaccine compared to patients not receiving the vaccine. These IgG antibodies can be specific for the MERS-CoV antigen. These IgG antibodies can be reactive with the MERS-CoV antigen. Preferably, the humoral response is cross-reactive with two or more of the MERS-CoV strains. The IgG antibody concentration of the patient receiving the vaccine is about 1.5 times to about 16 times, about 2 times to about 12 times, or about 3 times to about 10 times that of a patient not receiving the vaccine. Can be increased. The IgG antibody concentration of the patient receiving the vaccine is at least about 1.5 times, at least about 2.0 times, at least about 2.5 times, at least about 3.0, compared to patients not receiving the vaccine. Times, at least about 3.5 times, at least about 4.0 times, at least about 4.5 times, at least about 5.0 times, at least about 5.5 times, at least about 6.0 times, at least about 6.5 times At least about 7.0 times, at least about 7.5 times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, at least about 9.5 times, at least about 10.0 times, At least about 10.5 times, at least about 11.0 times, at least about 11.5 times, at least about 12.0 times, at least about 12.5 times, at least about 13.0 times, at least about 13.5 times, at least About 1 .0 fold, at least about 14.5-fold, at least about 15.0-fold, at least about 15.5 fold, or may increase to at least about 16.0 times.

The vaccine can induce a cellular immune response in patients receiving the vaccine. The induced cellular immune response can be specific for the MERS-CoV antigen. The induced cellular immune response can be reactive with the MERS-CoV antigen. Preferably, the cellular response is cross-reactive with two or more of the MERS-CoV strains. The induced cellular immune response can include eliciting a CD8 ⁺ T cell response. The induced CD8 ⁺ T cell response can be reactive with the MERS-CoV antigen. The induced CD8 ⁺ T cell response can be multifunctional. The induced cellular immune response can include the induction of a CD8 ⁺ T cell response, wherein the CD8 ⁺ T cells are interferon-gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), interleukin -2 (IL-2), or a combination of IFN-γ and TNF-α.

The induced cellular immune response is related to the CD8 of patients receiving the vaccine compared to patients not receiving the vaccine. ⁺ May include increased T cell response. The CD8 of the patient receiving the vaccine ⁺ The T cell response can be increased from about 2-fold to about 30-fold, from about 3-fold to about 25-fold, or from about 4-fold to about 20-fold compared to patients not receiving the vaccine. The CD8 of the patient receiving the vaccine ⁺ T cell response is at least about 1.5 times, at least about 2.0 times, at least about 3.0 times, at least about 4.0 times, at least about 5.0 times compared to patients not receiving the vaccine At least about 6.0 times, at least about 6.5 times, at least about 7.0 times, at least about 7.5 times, at least about 8.0 times, at least about 8.5 times, at least about 9.0 times, At least about 9.5 times, at least about 10.0 times, at least about 10.5 times, at least about 11.0 times, at least about 11.5 times, at least about 12.0 times, at least about 12.5 times, at least About 13.0, at least about 13.5, at least about 14.0, at least about 14.5, at least about 15.0, at least about 16.0, at least about 17.0, at least about 1 0.0 times, at least about 19.0 times, at least about 20.0 times, at least about 21.0 times, at least about 22.0 times, at least about 23.0 times, at least about 24.0 times, at least about 25. It can be increased to 0, at least about 26.0, at least about 27.0, at least about 28.0, at least about 29.0, or at least about 30.0.

The induced cellular immune response can include an increased frequency of CD3 ⁺ CD8 ⁺ T cells that produce IFN-γ. The frequency of the CD3 ⁺ CD8 ⁺ IFN-γ ⁺ T cells in patients receiving the vaccine is at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold compared to patients not receiving the vaccine 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times.

The induced cellular immune response can include an increased frequency of CD3 ⁺ CD8 ⁺ T cells that produce TNF-α. The frequency of the CD3 ⁺ CD8 ⁺ TNF-α ⁺ T cells in patients receiving the vaccine is at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold compared to patients not receiving the vaccine , 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, or 14 times.

The induced cellular immune response may include an increased frequency of CD3 ⁺ CD8 ⁺ T cells that produce IL-2. The frequency of the CD3 ⁺ CD8 ⁺ IL-2 ⁺ T cells in patients receiving the vaccine is at least about 0.5, 1.0, 1.5 times compared to patients not receiving the vaccine 2.0 times, 2.5 times, 3.0 times, 3.5 times, 4.0 times, 4.5 times, or 5.0 times.

The induced cellular immune response can include an increased frequency of CD3 ⁺ CD8 ⁺ T cells that produce both IFN-γ and TNF-α. The frequency of the CD3 ⁺ CD8 ⁺ IFN-γ ⁺ TNF-α ⁺ T cells in patients receiving the vaccine is at least about 25-fold, 30-fold, 35-fold, 40-fold compared to patients not receiving the vaccine. Times, 45 times, 50 times, 55 times, 60 times, 65 times, 70 times, 75 times, 80 times, 85 times, 90 times, 95 times, 100 times, 110 times, 120 times, 130 times, 140 times, It can be increased up to 150 times, 160 times, 170 times, or 180 times.

The cellular immune response induced with the vaccine may include the induction of a CD4 ⁺ T cell response. The induced CD4 ⁺ T cell response can be reactive with the MERS-CoV antigen. The induced CD4 ⁺ T cell response can be multifunctional. The induced cellular immune response can include the induction of a CD4 ⁺ T cell response, and the CD4 ⁺ T cells are IFN-γ, TNF-α, IL-2, or IFN-γ and TNF-α. Produce a combination.

The induced cellular immune response may include an increased frequency of CD3 ⁺ CD4 ⁺ T cells that produce IFN-γ. The frequency of the CD3 ⁺ CD4 ⁺ IFN-γ ⁺ T cells in patients receiving the vaccine is at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold compared to patients not receiving the vaccine 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, or 20 times.

The induced cellular immune response may include an increased frequency of CD3 ⁺ CD4 ⁺ T cells that produce TNF-α. The frequency of the CD3 ⁺ CD4 ⁺ TNF-α ⁺ T cells in patients receiving the vaccine is at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold compared to patients not receiving the vaccine 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, or up to 22 times May increase.

The induced cellular immune response can include an increased frequency of CD3 ⁺ CD4 ⁺ T cells that produce IL-2. The frequency of the CD3 ⁺ CD4 ⁺ IL-2 ⁺ T cells in patients receiving the vaccine is at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold compared to patients not administered the vaccine 7 times 8 times 9 times 10 times 11 times 12 times 13 times 14 times 15 times 16 times 17 times 18 times 19 times 20 times 21 times 22 times 23 times Times, 24 times, 25 times, 26 times, 27 times, 28 times, 29 times, 30 times, 31 times, 32 times, 33 times, 34 times, 35 times, 36 times, 37 times, 38 times, 39 times, It can be increased up to 40 times, 45 times, 50 times, 55 times, or 60 times.

The induced cellular immune response may include an increase in the frequency of CD3 ⁺ CD4 ⁺ T cells that produce both IFN-γ and TNF-α. The frequency of the CD3 ⁺ CD4 ⁺ IFN-γ ⁺ TNF-α ⁺ in patients receiving the vaccine is at least about 2-fold, 2.5-fold, 3.0-fold compared to patients not receiving the vaccine 3.5 times, 4.0 times, 4.5 times, 5.0 times, 5.5 times, 6.0 times, 6.5 times, 7.0 times, 7.5 times, 8.0 times 8.5 times, 9.0 times, 9.5 times, 10.0 times, 10.5 times, 11.0 times, 11.5 times, 12.0 times, 12.5 times, 13.0 times 13.5 times, 14.0 times, 14.5 times, 15.0 times, 15.5 times, 16.0 times, 16.5 times, 17.0 times, 17.5 times, 18.0 times 18.5 times, 19.0 times, 19.5 times, 20.0 times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times, 28 times, 29 times, 30 times , 31 times, 32 times, 33 times, 34 times, or up to 35 times It can be pressurized.

  The vaccine of the present invention can be provided with characteristics required for an effective vaccine that is safe and the vaccine itself does not cause disease or death; for diseases caused by exposure to live pathogens such as viruses and bacteria. Prevent; induce neutralizing antibodies to prevent cellular invasion; induce protective T cells against intracellular pathogens; in addition, ease of administration, fewer side effects, biological stability, and per dose Provide low cost.

  The vaccine can further induce an immune response when administered to different tissues such as muscle and skin. The vaccine may further induce an immune response upon administration by electroporation, infusion, subcutaneous injection, or intramuscular injection.

a. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Antigen As noted above, the vaccine comprises a MERS-CoV antigen, a fragment thereof, a variant thereof, or a combination thereof. Coronaviruses, including MERS-CoV, have a type 1 membrane glycoprotein known as a spike (S) protein that is encapsulated in a membrane and forms a protruding spike on the surface of the coronavirus. The spike protein facilitates binding of the coronavirus to a protein located on the cell surface, such as metalloprotease aminopeptidase N, and mediates cell-virus membrane fusion. Specifically, the spike protein includes an S1 subunit that facilitates binding of the coronavirus to a cell surface protein. Thus, the S1 subunit of the spike protein controls which cells are infected with the coronavirus. The spike protein also includes the S2 subunit, which is a transmembrane subunit that facilitates fusion of the virus and cell membranes. Thus, the MERS-CoV antigen can comprise the MERS-CoV spike protein, the S1 subunit of the MERS-CoV spike protein, or the S2 subunit of the MERS-CoV spike protein.

  After binding to cell surface proteins and membrane fusion, the coronavirus enters the cell and its single-stranded RNA genome is released into the cytoplasm of the infected cell. The single stranded RNA genome is positive and can therefore be translated into an RNA polymerase that produces additional viral RNA that is negative. Thus, the MERS-CoV antigen can also be a MERS-CoV RNA polymerase.

  The minus RNA strand of the virus is transcribed into smaller, subgenomic plus RNA strands, which translate into other viral proteins such as nucleocapsid (N) protein, envelope (E) protein, and matrix (M) protein. Used. Thus, the MERS-CoV antigen may comprise a MERS-CoV nucleocapsid protein, a MERS-CoV envelope protein, or a MERS-CoV matrix protein.

  The negative RNA strand of the virus can also be used for replication of the viral genome linked by a nucleocapsid protein. Matrix proteins are incorporated into the endoplasmic reticulum of infected cells along with spike proteins. At the same time, the nucleocapsid protein bound to the viral genome and the matrix and spike proteins embedded in the membrane bud into the lumen of the endoplasmic reticulum, thereby enveloping the viral genome with the membrane. The progeny of the virus is then transported by the Golgi vesicles to the cell membrane of the infected cell and released into the extracellular space by endocytosis.

  In some embodiments, the MERS-CoV antigen comprises MERS-CoV spike protein, MERS-CoV RNA polymerase, MERS-CoV nucleocapsid protein, MERS-CoV envelope protein, MERS-CoV matrix protein, fragments thereof, these It can be a variant or a combination thereof. The MERS-CoV antigen comprises 2 or more MERS-CoV spike antigens, 2 or more MERS-CoV RNA polymerases, 2 or more MERS-CoV nucleocapsid proteins, 2 or more envelope proteins, 2 or It can be a consensus antigen from further matrix proteins, or combinations thereof. The MERS-CoV consensus antigen may be modified for good expression. Modifications may include increasing the immunogenicity of the MERS-CoV antigen by codon optimization, RNA optimization, adding Kozak sequences to advance translation initiation, and / or adding immunoglobulin leader sequences . In some embodiments, the MERS-CoV antigen can be the amino acid sequence set forth in SEQ ID NO: 6 and comprises an IgE leader encoded by the nucleotide sequence set forth in SEQ ID NO: 5.

(1) MERS-CoV spike antigen The MERS-CoV antigen may be a MERS-CoV spike antigen, a fragment thereof, a variant thereof, or a combination thereof. The MERS-CoV spike antigen is capable of eliciting a mammalian immune response against one or more MERS-CoV strains. The MERS-CoV spike antigen may comprise an epitope that is particularly effective as an immunogen from which an anti-MERS-CoV immune response can be induced.

  The MERS-CoV spike antigen can be a consensus sequence from two or more MERS-CoV strains. The MERS-CoV spike antigen may include consensus sequences and / or modifications for good expression. Modifications include increasing the immunogenicity of the MERS-CoV spike antigen by codon optimization, RNA optimization, the addition of a Kozak sequence to advance translation initiation, and / or the addition of an immunoglobulin leader sequence. sell. The MERS-CoV consensus spike antigen may comprise an immunoglobulin signal peptide, for example, a signal peptide such as, but not limited to, immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. In some embodiments, the MERS-CoV consensus spike antigen may include a hemagglutinin (HA) tag. The MERS-CoV consensus spike antigen can be designed to elicit a stronger and broader cellular and / or humoral immune response than the corresponding codon optimized spike antigen.

  The MERS-CoV consensus spike antigen can be the nucleic acid sequence SEQ ID NO: 1 encoding SEQ ID NO: 2 (FIGS. 8A and 8B). In some embodiments, the MERS-CoV consensus spike antigen is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86% of the total length of the nucleic acid sequence set forth in SEQ ID NO: 1. The nucleic acid sequence can be 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous. In other embodiments, the MERS-CoV consensus spike antigen is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87 of the full length amino acid sequence set forth in SEQ ID NO: 2. %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence encoding a homologous amino acid sequence It can be.

  The MERS-CoV consensus spike antigen can be amino acid sequence SEQ ID NO: 2. In some embodiments, the MERS-CoV consensus spike antigen is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86% of the full length amino acid sequence set forth in SEQ ID NO: 2. The amino acid sequence can be 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% homologous.

  An immunogenic fragment of SEQ ID NO: 2 may be provided. The immunogenic fragment is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% of SEQ ID NO: 2, It may comprise at least 98%, or at least 99%. In some embodiments, the immunogenic fragment comprises a leader sequence, such as an immunoglobulin leader such as an IgE leader. In some embodiments, the immunogenic fragment does not include a leader sequence.

  An immunogenic fragment of a protein of amino acid sequence homologous to the immunogenic fragment of SEQ ID NO: 2 may be provided. Such immunogenic fragments are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of a protein that is 95% homologous to SEQ ID NO: 2. It may comprise at least 96%, at least 97%, at least 98%, or at least 99%. Some embodiments relate to immunogenic fragments that are 96% homologous to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments that are 97% homologous to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments that are 98% homologous to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments that are 99% homologous to the immunogenic fragments of the consensus protein sequences herein. In some embodiments, the immunogenic fragment comprises a leader sequence, such as an immunoglobulin leader such as an IgE leader. In some embodiments, the immunogenic fragment does not include a leader sequence.

  Some embodiments relate to immunogenic fragments of SEQ ID NO: 1. The immunogenic fragment is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% of SEQ ID NO: 1. , At least 98%, or at least 99%. The immunogenic fragment can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragment of SEQ ID NO: 1. In some embodiments, the immunogenic fragment comprises a sequence encoding a leader sequence, such as an immunoglobulin leader such as an IgE leader. In some embodiments, the fragment does not include a coding sequence that encodes a leader sequence.

(A) Cytoplasmic domain-deficient MERS-CoV spike antigen The MERS-CoV antigen is a cytoplasmic domain-deficient MERS-CoV spike antigen (also referred to herein as “MERS-CoV spike antigen ΔCD”), a fragment thereof, a variant thereof, or a It can be a combination. The MERS-CoV spike antigen ΔCD is capable of eliciting a mammalian immune response against one or more MERS-CoV strains. The MERS-CoV spike antigen ΔCD may comprise an epitope that is particularly effective as an immunogen from which an anti-MERS-CoV immune response can be induced.

  The MERS-CoV spike antigen ΔCD can be a consensus sequence from two or more MERS-CoV strains. The MERS-CoV spike antigen ΔCD may include consensus sequences and / or modifications for good expression. Modifications may increase the immunogenicity of the MERS-CoV spike antigen ΔCD by codon optimization, RNA optimization, the addition of a Kozak sequence to advance translation initiation, and / or the addition of an immunoglobulin leader sequence. May be included. The MERS-CoV consensus spike antigen ΔCD may comprise an immunoglobulin signal peptide, for example, a signal peptide such as, but not limited to, immunoglobulin E (IgE) or immunoglobulin (IgG) signal peptide. In some embodiments, the consensus spike antigen ΔCD can comprise a hemagglutinin (HA) tag. The MERS-CoV consensus spike antigen ΔCD can be designed to elicit stronger and broader cellular and / or humoral immune responses than the corresponding codon optimized spike antigen ΔCD.

  The MERS-CoV consensus spike antigen ΔCD can be the nucleic acid sequence SEQ ID NO: 3 encoding SEQ ID NO: 4 (FIGS. 8C and 8D). In some embodiments, the MERS-CoV consensus spike antigen ΔCD is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96% of the total length of the nucleic acid sequence set forth in SEQ ID NO: 3. 97%, 98%, 99%, or 100% homologous nucleic acid sequences. In other embodiments, the MERS-CoV consensus spike antigen ΔCD is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96% of the full length amino acid sequence set forth in SEQ ID NO: 4, It can be a nucleic acid sequence encoding an amino acid sequence that is 97%, 98%, 99%, or 100% homologous.

  The MERS-CoV consensus spike antigen ΔCD can be amino acid sequence SEQ ID NO: 4. In some embodiments, the MERS-CoV consensus spike antigen ΔCD is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96% of the full length of the amino acid sequence set forth in SEQ ID NO: 4. 97%, 98%, 99%, or 100% homologous amino acid sequences.

  An immunogenic fragment of SEQ ID NO: 4 may be provided. The immunogenic fragment is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% of SEQ ID NO: 4 , At least 98%, or at least 99%. In some embodiments, the immunogenic fragment comprises a leader sequence, such as an immunoglobulin leader such as an IgE leader. In some embodiments, the immunogenic fragment does not include a leader sequence.

  An immunogenic fragment of a protein of amino acid sequence homologous to the immunogenic fragment of SEQ ID NO: 4 may be provided. Such immunogenic fragments are at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of a protein that is 95% homologous to SEQ ID NO: 4, It may comprise at least 96%, at least 97%, at least 98%, or at least 99%. Some embodiments relate to immunogenic fragments that are 96% homologous to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments that are 97% homologous to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments that are 98% homologous to the immunogenic fragments of the consensus protein sequences herein. Some embodiments relate to immunogenic fragments that are 99% homologous to the immunogenic fragments of the consensus protein sequences herein. In some embodiments, the immunogenic fragment comprises a leader sequence, such as an immunoglobulin leader such as an IgE leader. In some embodiments, the immunogenic fragment does not include a leader sequence.

  Some embodiments relate to immunogenic fragments of SEQ ID NO: 3. The immunogenic fragment is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% of SEQ ID NO: 3, It can be at least 98%, or at least 99%. The immunogenic fragment can be at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the fragment of SEQ ID NO: 3. In some embodiments, the immunogenic fragment comprises a sequence encoding a leader sequence, such as an immunoglobulin leader such as an IgE leader. In some embodiments, the fragment does not include a coding sequence that encodes a leader sequence.

b. Vector The vaccine may comprise one or more vectors comprising a nucleic acid encoding the antigen. The one or more vectors may be capable of expressing the antigen. The vector may have a nucleic acid sequence that includes an origin of replication. The vector can be a plasmid, bacteriophage, bacterial artificial chromosome, or yeast artificial chromosome. The vector can be either a self-replicating extrachromosomal vector or a vector that coalesces with the host genome.

  The one or more vectors may be expression constructs that are typically plasmids used to introduce specific genes into target cells. When the expression vector is in a cell, the protein encoded by the gene is produced by the ribosome complex of the cellular transcription and translation machinery. The plasmids are often engineered to contain regulatory sequences that act as enhancer and promoter regions, resulting in efficient transcription of the gene on the expression vector. The vectors of the present invention express large amounts of stable messenger RNA and thus proteins.

  The vector may comprise a strong promoter, a strong stop codon, regulation of the distance between the promoter and the cloned gene, and expression signals such as insertion of a transcription termination sequence and PTIS (Portable Translation Initiation Sequence).

(1) Expression vector The vector may be a circular plasmid or a linear nucleic acid. The circular plasmid and linear nucleic acid can direct the expression of a specific nucleotide sequence in the appropriate patient cell. The vector may have a promoter operably linked to a nucleotide sequence that encodes the antigen and may be operably linked to a termination signal. The vector may also contain sequences necessary for proper translation of the nucleotide sequence. The vector comprising the nucleotide sequence of interest may be a chimera, meaning that at least one of its components is heterologous to at least one other component. Expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to some specific external stimulus. In the case of multicellular organisms, the promoter may also be specific for a particular tissue, organ, or developmental stage.

(2) Circular and linear vectors The vector may be a circular plasmid, which either transforms the target cell by fusion to the cell genome or exists extrachromosomally (eg, self-replicating with an origin of replication. Plasmid).

  The vector can be pVAX, pcDNA3.0, or provax, or any other expression vector capable of expressing DNA that encodes the antigen and allows the cell to translate the sequence into an antigen recognized by the immune system It's okay.

  Also provided herein is a linear nucleic acid vaccine, or linear expression cassette (“LEC”), that can be efficiently delivered to a patient by electroporation and that expresses one or more desired antigens. The LEC may be any linear DNA lacking any phosphate backbone. The DNA can encode one or more antigens. The LEC may include a promoter, intron, stop codon, and / or polyadenylation signal. The expression of the antigen can be controlled by the promoter. The LEC may not have any antibiotic resistance gene and / or phosphate backbone. The LEC may not have other nucleic acid sequences that are not related to the desired antigen gene expression.

  The LEC may be derived from any plasmid that can be linearized. The plasmid may be capable of expressing the antigen. The plasmid can be pNP (Puerto Rico / 34) or pM2 (New Caledonia / 99). The plasmid can be WLV009, pVAX, pcDNA3.0, or provax, or any other capable of expressing DNA that encodes the antigen and allows the cell to translate the sequence into an antigen recognized by the immune system. It may be an expression vector.

  The LEC can be pcrM2. The LEC can be pcrNP. pcrNP and pcrMR can be derived from pNP (Puerto Rico / 34) and pM2 (New Caledonia / 99), respectively.

(3) Promoter, intron, stop codon, and polyadenylation signal The vector may have a promoter. The promoter can be any promoter capable of manipulating gene expression and regulating the expression of isolated nucleic acids. Such promoters are cis-acting sequence elements required for transcription via DNA-dependent RNA polymerase that transcribes the antigen sequences described herein. The choice of promoter used to direct the expression of heterologous nucleic acid depends on the particular application. The promoter may be located in the vector at a distance from the start of transcription that is approximately the same distance from the transcription start site in the natural environment. However, this variation in distance can be adjusted without compromising the function of the promoter.

  The promoter may be operably linked to the nucleic acid sequence encoding the antigen and signals necessary for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. The promoter has been shown to be effective for expression in CMV promoter, SV40 early promoter, SV40 late promoter, metallothionein promoter, mouse breast cancer virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or eukaryotic cells. Another promoter may be used.

  The vector may contain enhancers and introns along with functional splice donor and acceptor sites. The vector may include a transcription termination region downstream of the structural gene to provide efficient termination. The termination region may be obtained from the same gene as the promoter sequence or from a different gene.

c. Vaccine excipients and other components The vaccine may further comprise pharmaceutically acceptable excipients. The pharmaceutically acceptable excipient can be a functional molecule such as a solvent, carrier, or diluent. The pharmaceutically acceptable excipients include surfactants such as immunostimulatory complex (ISCOMS), incomplete Freund's adjuvant, LPS analogues including monophosphoryl lipid A, muramyl peptides, quinone analogues, squalene and squalene. Transfection facilitators that may include vesicles such as hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions, polycations, or nanoparticles, or other well known transfection facilitators.

  The transfection facilitating agent is a polyanion, polycation, or lipid, including poly-L-glutamic acid (LGS). The transfection facilitating agent is poly-L-glutamic acid, and the poly-L-glutamic acid may be present in the vaccine at a concentration of less than 6 mg / ml. The transfection facilitating agent is a surfactant such as an immunostimulatory complex (ISCOMS), incomplete Freund's adjuvant, LPS analog containing monophosphoryl lipid A, muramyl peptide, quinone analog, and vesicles such as squalene and squalene. Hyaluronic acid may also be administered with the gene construct. The DNA plasmid vaccine is a liposome, calcium ion, viral protein, polyanion, polycation, or nanoparticle, including other liposomes well known in the art, such as lipids, lecithin liposomes or DNA-liposome mixtures (see eg W09324640). Or other well known transfection facilitating agents may also be included. The transfection facilitating agent is a polyanion, polycation, or lipid, including poly-L-glutamic acid (LGS). The concentration of the transfection agent in the vaccine is less than 4 mg / ml, less than 2 mg / ml, less than 1 mg / ml, less than 0.750 mg / ml, less than 0.500 mg / ml, less than 0.250 mg / ml, 0.100 mg / Ml, less than 0.050 mg / ml, or less than 0.010 mg / ml.

  The pharmaceutically acceptable excipient can be an adjuvant. The adjuvant can be another gene expressed in another plasmid or given as a protein to the vaccine in combination with the plasmid. The adjuvant includes α-interferon (IFN-α), β-interferon (IFN-β), γ-interferon, platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), Skin T cell attracting chemokine (CTACK), chemokine expressed in thymic epithelial cells (TECK), mucosa-associated epithelial chemokine (MEC), IL-12, IL-15, MHC, CD80, CD86 Can be selected from the group consisting of IL-15 containing a signal peptide from IgE. The adjuvant is IL-12, IL-15, IL-28, CTACK, TECK, platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), IL-1, IL- 2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-18, or combinations thereof.

  Other genes that may be useful as adjuvants are: MCP-1, MIP-1a, MIP-1p, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM-1, MadCAM- 1, LFA-1, VLA-1, Mac-1, p150.95, PECAM, ICAM-1, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, IL-8 mutant type, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, IL-22, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Flt, Apo- 1, p55, WSL-1, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAI -R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-1, Ap-1, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-1, JNK, interferon responsive gene, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2G, MICA, MIGB, NKG2GK, NKG2GK , TAP1, TAP2, and genes encoding these functional fragments.

  The vaccine is further described in U.S. application filed Apr. 1, 1994, which is incorporated by reference in its entirety. S. Serial No. The gene vaccine promoter described in 021,579 may be included.

  The vaccine can be formulated depending on the method of administration used. The pharmaceutical composition of an injectable vaccine can be sterile, pyrogen-free, and microparticle-free. Isotonic formulations or solutions can be used. Examples of isotonic additives include sodium chloride, dextrose, mannitol, sorbitol, and lactose. The vaccine can include a vasoconstrictor. The isotonic solution can include phosphate buffered saline. The vaccine may further comprise stabilizers including gelatin and albumin. The stabilizer stabilizes the formulation containing LGS or polycation or polyanion for a long period of time at room temperature or ambient temperature.

3. Methods of vaccination Also disclosed herein are methods of treating, preventing, and / or protecting against disease in a patient in need of treatment, prevention, and / or protection from the disease by administering the vaccine to the patient. provide. Administration of the vaccine to the patient can induce or elicit an immune response in the patient. The induced immune response can be utilized to treat, prevent, and / or protect against diseases, eg, lesions associated with MERS-CoV infection. The induced immune response conferred resistance to one or more MERS-CoV strains in patients receiving the vaccine.

The induced immune response can induce an induced humoral immune response and / or an induced cellular immune response. The humoral immune response can be induced from about 1.5 times to about 16 times, from about 2 times to about 12 times, or from about 3 times to about 10 times. The induced humoral immune response can include IgG antibodies and / or neutralizing antibodies reactive with the antigen. The induced cellular immune response can induce a CD8 ⁺ T cell response induced from about 2-fold to about 30-fold, from about 3-fold to about 25-fold, or from about 4-fold to about 20-fold.

  The dose of the vaccine may be 1 μg to 10 mg active ingredient / kg body weight / hour, or 20 μg to 10 mg ingredient / kg body weight / hour. The vaccine is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, It may be administered every 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of vaccine doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

a. Administration The vaccine can be formulated according to standard techniques well known to pharmaceutical practitioners. Such compositions can be administered at dosages and techniques well known to medical practitioners, taking into account factors such as the age, sex, weight, and condition of the individual patient and the method of administration. The patient can be a mammal, such as a human, horse, cow, pig, sheep, cat, dog, rat, or mouse.

  The vaccine can be administered prophylactically or therapeutically. For prophylactic administration, the vaccine may be administered in an amount sufficient to induce an immune response. For therapeutic applications, the vaccine is administered in an amount sufficient to induce a therapeutic effect in a patient in need thereof. An amount adequate to accomplish this is defined as “therapeutically effective dose”. Effective amounts for this use will depend, for example, on the particular composition of the vaccine regimen administered, the method of administration, the stage and severity of the disease, the normal health of the patient, and the judgment of the prescribing physician.

  The vaccine is described in Donnelly et al. (Ann. Rev. Immunol. 15: 617-648 (1997)); Felgner et al. (US Pat. No. 5,580), which is incorporated herein by reference in its entirety. , 859, issued December 3, 1996); Felgner (US Pat. No. 5,703,055, issued December 30, 1997); and Carson et al. (US Pat. No. 5). , 679, 647, published October 21, 1997), and can be administered by methods well known to those skilled in the art. The DNA of the vaccine can be conjugated to particles or beads that can be administered to an individual using, for example, a vaccine gun. One skilled in the art will appreciate that the selection of a pharmaceutically acceptable carrier containing a physiologically acceptable compound will depend, for example, on the method of administration of the expression vector.

  The vaccine can be supplied in a variety of ways. Typical delivery methods include parenteral administration, such as intradermal, intramuscular, or subcutaneous delivery. Other methods include oral administration, intranasal, and intravaginal routes. In particular, for the DNA of the vaccine, the vaccine can be supplied into the interstitial space of individual tissues (Felner et al., US Pat. No. 5,580,859 and 5,703,055, all of these contents) Are incorporated herein by reference in their entirety). The vaccine can also be administered intramuscularly, or can be administered intradermally or subcutaneously, or transdermally, eg, by iontophoresis. Epidermal administration of the vaccine can also be utilized. Epidermal administration may involve mechanically or chemically stimulating the outermost layer of the epidermis and stimulating an immune response to the stimulant (Carson et al., US Pat. No. 5,679,647, The entire contents are incorporated herein by reference).

  The vaccine can also be formulated for administration via the nasal cavity. Formulations suitable for nasal administration, where the carrier is a solid, may comprise a coarse powder having a particle size of, for example, from about 10 to about 500 microns, which is a method of inhaling through the nose, i.e. of the powder close to the nose. Administered by rapid inhalation from the container through the nasal cavity. The formulation may be a nasal spray or nasal spray, or by aerosol administration with a nebulizer. The formulation can include an aqueous or oily solution of the vaccine.

  The vaccine can be a liquid formulation such as a suspension, syrup, or elixir. The vaccine may be a formulation for parenteral, subcutaneous, intradermal, intramuscular, or intravenous administration (eg, injection administration), such as a sterile suspension or emulsion.

  The vaccine may be incorporated into liposomes, microspheres, or other polymer matrices (Felner et al., US Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vols. Ito III (2nd ed 1993), the contents of which are incorporated herein by reference in their entirety). Liposomes may consist of phospholipids or other lipids and may be non-toxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

  The vaccine is electroporated, such as U.S. Pat. S. Patent No. Administered by the method described in 7,664,545. The electroporation is a U.S. patent that is incorporated herein by reference in its entirety. S. Patent No. 6,302,874; 5,676,646; 6,241,701; 6,233,482; 6,216,034; 6,208,893; 6,192,270; 6,181,964; 150, 148; 6,120,493; 6,096,020; 6,068,650; and 5,702,359 can be used. The electroporation can be performed with a less invasive device.

  The less invasive electroporation device (“MID”) may be a device for injecting the vaccine and associated fluid into body tissue. The device may comprise a hollow needle, a DNA cassette, and a fluid supply means, and the device simultaneously (eg automatically) injects DNA into the body tissue during the insertion of the needle into the body tissue. As such, the fluid supply means is configured to be activated during use. This has the advantage that the ability to gradually inject the DNA and associated fluid at the same time the needle is inserted leads to a more uniform distribution of the fluid through the body tissue. The pain felt during the injection process can be attenuated due to the wider distribution of the injected DNA.

  The MID can inject the vaccine into the tissue without using a needle. The MID may inject the vaccine as a small stream or jet with a force such that the vaccine penetrates the surface of the tissue and enters the underlying tissue and / or muscle. The force behind the small flow or jet can be brought about by instantaneous expansion through a micro-orifice of compressed gas such as carbon dioxide. Examples of less invasive electroporation devices and methods for their use are disclosed in published U.S. Pat. S. Patent Application No. 200802234655; S. Patent No. 6,520,950; S. Patent No. 7,171,264; S. Patent No. 6, 208, 893; S. Patent NO. 6,009,347; S. Patent No. 6, 120, 493; S. Patent No. 7, 245, 963; S. Patent No. 7,328,064; S. Patent No. 6,763,264.

  The MID may comprise a syringe that causes a high velocity liquid jet to penetrate the tissue without pain. Such needleless injectors are commercially available. Examples of needleless injectors that can be used in this document include U.S. Pat. S. Patent No. 3,805,783; 4,447,223; 5,505,697; and 4,342,310.

  The desired vaccine in a form suitable for direct or indirect electrotransport is sufficient to use a needleless injector, usually bringing the surface of the tissue into contact with the injector, causing the penetration of the vaccine into the tissue. By actuating the supply of the drug jet with sufficient force, it can be introduced (eg, injected) into the tissue to be treated. For example, if the tissue to be treated is mucosa, skin, or muscle, the drug is directed toward the mucosa or skin surface, penetrating the drug through the stratum corneum, into the cortex, or underlying tissue and Fired with enough force to penetrate the muscles.

  Needleless injectors are well suited for delivering vaccines to all types of tissues, particularly skin and mucous membranes. In some embodiments, a needleless injector can be used to propel a liquid containing the vaccine toward the surface and into the patient's skin or mucous membrane. Representative examples of the various types of tissue that can be treated using the methods of the present invention include pancreas, larynx, nasopharynx, hypopharynx, oropharynx, lips, throat, lung, heart, kidney, muscle, chest, colon, Prostate, thymus, testis, skin, mucosal tissue, ovary, blood vessel, or any combination thereof.

  The MID may comprise a needle electrode that electroporates the tissue. For example, by emitting a pulse between a plurality of pairs of electrodes of a plurality of electrode arrays arranged in a rectangular or square pattern, a better result can be obtained than when a pulse is emitted between a pair of electrodes. For example, U.S. Pat. S. Patent No. 5,702,359, entitled “Needle Electrodes for Mediated Delivery of Drugs and Genes”, is an array of needles, where multiple pairs of needles can be pulsed during the course of therapeutic treatment . In its application, which is incorporated herein by reference as fully described, the needles are arranged in a circular array, but with a connector and a switch device that allow a pulse to be applied between a pair of opposing needle electrodes. Yes. A pair of needle electrodes for supplying the recombinant expression vector to the cells may be used. Such devices and systems are described in U.S. Pat. S. Patent No. 6,763,264, the contents of which are incorporated herein by reference. Alternatively, DNA injection and electroporation with a single needle, similar to a normal needle, can be pulsed at a lower voltage than that provided by currently used devices, thereby causing the patient to feel electricity. A single needle device that reduces the feeling of running may be used.

  The MID may include one or more electrode arrays. The array can include two or more needles of the same diameter or different diameters. The needles may be spaced evenly or unevenly. The needle may be 0.005 inches to 0.03 inches, 0.01 inches to 0.025 inches, or 0.015 inches to 0.020 inches. The needle may be 0.0175 inches in diameter. The needles may be spaced at intervals of 0.5 mm, 1.0 mm, 1.5 mm, 2.0 mm, 2.5 mm, 3.0 mm, 3.5 mm, 4.0 mm, or more.

  The MID may consist of a pulse generator and two or more needle vaccine injectors that deliver vaccine and electroporation pulses in one step. The pulse generator can provide flexible programming of pulses and infusion parameters by a personal computer operated with a flash card and comprehensive recording and storage of electroporation and patient data. The pulse generator can supply pulses of various volts in a short time. For example, the pulse generator may provide three 15 volt pulses with a duration of 100 ms. An example of such a MID is the Elgen 1000 system by Inovio Biomedical Corporation, which is a U.S. S. Patent No. 7,328,064, the contents of which are incorporated herein by reference.

  The MID may be a CELLECTRA (Inobio Pharmaceuticals, Bluebell, PA) device and system, which is a modular electrode system that introduces macromolecules such as DNA into cells of the body or selected tissues of plants. Facilitate. The modular electrode system may include a plurality of needle electrodes; an injection needle; an electrical connector that provides a conductive link from a programmable constant current pulse controller to the plurality of needle electrodes; and a power source. The operator can grab the plurality of needle electrodes mounted on the support structure and securely insert them into selected tissues of the body or plant. The polymer is then delivered to the selected tissue from the needle. The programmable constant current pulse controller is activated and a constant current electrical pulse is applied to the plurality of needle electrodes. The applied constant current electrical pulse facilitates introduction of the polymer into cells between the plurality of electrodes. Cell death due to cell overheating is minimized by limiting power loss in the tissue by constant current pulses. The Cellectra apparatus and system are described in U.S. Pat. S. Patent No. 7, 245, 963, the contents of which are incorporated herein by reference.

  The MID may be an Elgen 1000 system (Inobio Pharmaceuticals). The Elgen 1000 system may comprise a hollow needle; and a device providing a fluid supply means, the device being simultaneously (eg automatically) fluid in the course of insertion of the needle into body tissue, as described herein. In use, the fluid supply means is configured to be activated so that the vaccine is injected into the body tissue. The advantage is that the ability to gradually infuse the fluid as the needle is inserted leads to a more uniform distribution of the fluid through the body tissue. It is also conceivable that the pain felt during the injection process is attenuated due to the wider volume distribution of the injected fluid.

  Furthermore, the automatic infusion of fluid facilitates automatic monitoring and registration of the actual dose of infusion fluid. This data can be saved by the control device for documentation purposes as needed.

  It is understood that the infusion rate can be linear or non-linear, and that the infusion can be performed at the same time that the needle is further inserted into the body tissue after the needle has been inserted into the skin of the patient to be treated. Let's be done.

  Suitable tissues into which fluid can be injected by the device of the present invention include tumor tissue, skin, liver tissue, but may be muscle tissue.

  The device further includes needle insertion means for guiding the insertion of the needle into the body tissue. The fluid injection rate is controlled by the needle insertion rate. This has the advantage that both the needle insertion and fluid injection can be controlled so that the insertion rate is matched to the injection rate as desired. This also makes the device easier to operate for the user. If necessary, means for automatically inserting the needle into the body tissue may be provided.

  The user can select when to start fluid injection. Ideally, however, the infusion is initiated when the tip of the needle reaches the muscle tissue, and the device comprises means for sensing when the needle has been inserted to a depth sufficient to initiate infusion of the fluid. May be. This means that fluid injection can be instructed to automatically start when the needle reaches the desired depth (usually the depth at which muscle tissue begins). The depth at which muscle tissue begins can be considered a predetermined needle insertion depth, such as a value of 4 mm that would be considered sufficient for the needle to pass through the skin layer.

  The sensing means may include an ultrasound probe. The sensing means may include means for sensing a change in impedance or resistance. In this case, the means may not itself record the depth of the needle in the body tissue, but to sense changes in impedance or resistance as the needle moves from the heterogeneous body tissue to the muscle. It will be configured. Either of these options provides a sensing means by which infusion can begin, which is relatively accurate and simple to operate. The needle insertion depth is further recorded as needed and can be utilized to control fluid injection such that the volume of fluid to be injected is determined as the needle insertion depth is recorded.

  The apparatus can further include a base that supports the needle; and a housing that receives the base, the base being in a first rearward position relative to the housing. When stored in the housing and the base is in a second forward position within the housing, the needle is movable relative to the housing such that the needle extends from the housing. This is convenient for the user because the position of the housing can be adjusted with the patient's skin and the needle can then be inserted into the patient's skin by moving the housing relative to the base.

  As mentioned above, it is desirable to obtain a controlled fluid infusion rate so that fluid is evenly distributed over the length of the needle as it is inserted into the skin. The fluid providing means may include piston drive means configured to inject fluid at a controlled rate. The piston drive means can be actuated by a servo motor, for example. However, the piston drive means may be actuated by the base moved axially relative to the housing. It will be appreciated that alternative means of fluid supply can be provided. Thus, for example, a sealed container that can be crushed can be provided instead of a syringe and piston system for fluid supply at a controlled or uncontrolled rate.

  The above device can be used for any kind of injection. However, since this is assumed to be particularly useful in the field of electroporation, it may further include means for applying a voltage to the needle. This allows the needle to be used not only for injection but also as an electrode in the process of electroporation. This is particularly advantageous because it means that an electric field is applied to the same site as the injected fluid. Because it is very difficult to accurately match the previously injected fluid with the electrode, the user will try to inject a larger volume of fluid into the larger site to ensure overlap between the injected material and the electric field, There is a traditional problem with electroporation that tends to apply an electric field over a larger area. By utilizing the present invention, both the fluid injection volume and the applied size of the electric field can be reduced while the electric field and the fluid are well matched.

4). Kits Provided herein are kits that can be used to treat patients using the vaccination methods described above. The kit can include the vaccine.

  The kit may also include instructions for performing the vaccination method and / or instructions for using the kit. The instructions included in the kit can be attached to the packaging material or included as a package insert. The description is usually written or printed, but is not limited thereto. Any medium capable of storing the description and communicating it to the ultimate user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (eg, magnetic disks, tapes, cartridges), optical media (eg, CD ROM), and the like. In this document, the term “description” may include the address of an Internet site that provides the description.

  The present invention has multiple aspects, exemplified by the following non-limiting examples.

5. Example Example 1
MERS-CoV Consensus Spike Antigen As described elsewhere in this document, there are multiple MERS-CoV strains as shown in Figure 3, and the consensus spike antigen explains the differences between these multiple MERS-CoV strains. Produced to do. Therefore, the MERS-CoV consensus spike antigen was obtained from the respective spike antigens of the viruses listed in FIG. A phylogenetic tree analysis based on the spike antigen was performed, and the MERS-CoV consensus spike antigen was listed in relation to its component spike antigen (FIG. 3, the label “MERS-HCoV-Consensus” is MERS-CoV consensus Showed spike antigen). The MERS-CoV consensus spike antigen also contained an N-terminal immunoglobulin E (IgE) leader sequence. The nucleic acid and amino acid sequences constituting the MERS-CoV consensus spike antigen are shown in FIGS. 8A and 8B, respectively. In FIG. 8A, the underlined portion indicates the nucleotide encoding the IgE leader sequence. In FIG. 8B, the underlined portion indicates the IgE leader sequence.

  A Kozak sequence is located at the 5 ′ end of the nucleic acid sequence encoding the MERS-CoV consensus spike antigen, and the resulting nucleic acid sequence is located between the BamHI and XhoI sites of the pVAX1 vector (Life Technologies, Carlsbad, Calif.). Inserted (FIG. 4A, the resulting construct was named MERS-HCoV-WT). The correct insertion of this nucleic acid sequence into the pVAX1 vector was confirmed by digestion with BamHI and XhoI followed by gel electrophoresis (FIG. 4B). In FIG. 4B, lane M shows the marker, lane 1 is the undigested MERS-HCoV-WT construct, and lane 2 is the digested MERS-HCoV-WT construct. Digestion results in the expected fragment size, and the MERS-HCoV-WT construct contains the pVAX1 vector and the inserted nucleic acid sequence (ie, the nucleic acid sequence comprising the Kozak sequence and encoding the MERS-CoV consensus spike antigen). Indicated that it contained.

  The structure and properties of this MERS-HCoV-WT construct are also described in Examples 9 and 10 below.

Example 2
Cytoplasmic domain-deficient MERS-CoV consensus spike antigen The spike antigen from coronavirus is a type 1 membrane glycoprotein with a transmembrane domain, which in turn determines the cytoplasmic and non-cytoplasmic domains of the spike antigen. In order to determine the position of the cytoplasmic domain in the MERS-CoV spike antigen, the position of the domain within the various MERS-CoV spike antigens was predicted using web software. Specifically, the web software Phobius and InterProScan were adopted for this analysis. Representative results from the Phobias and InterProScan analyzes are shown in FIGS. 5 and 6, respectively.

  From this domain analysis of the MERS-CoV spike antigen, a second MERS-CoV consensus spike antigen lacking the cytoplasmic domain was created. Specifically, the nucleic acid sequence encoding the MERS-CoV consensus spike antigen (described in Example 1, FIG. 8A, SEQ ID NO: 1) is modified and the translated protein does not contain the cytoplasmic domain (ie, MERS -Two stop codons were inserted as in (CoV consensus spike antigen ΔCD).

  The nucleic acid and amino acid sequences of the MERS-CoV consensus spike antigen ΔCD are shown in FIGS. 8C and 8D, respectively. In FIG. 8C, the underlined portion indicates the nucleotide encoding the IgE leader sequence, and the double underlined portion indicates the two inserted stop codons (prevented translation of the cytoplasmic domain compared to SEQ ID NO: 1). Indicates. In FIG. 8D, the underlined portion indicates the IgE leader sequence. A schematic diagram illustrating the resulting construct, MERS-HCoV-ΔCD, is shown in FIG. 7A.

  The MERS-HCoV-ΔCD construct was digested with BamHI and XhoI, followed by gel electrophoresis to confirm that the insert was present in the pVAX1 vector (FIG. 7B). In FIG. 7B, lane M shows the marker, lane 1 is the undigested MERS-HCoV-ΔCD construct and lane 2 is the digested MERS-HCoV-ΔCD construct. Digestion results in the expected fragment size and the MERS-HCoV-ΔCD construct is the pVAX1 vector and inserted nucleic acid sequence (ie, the nucleic acid sequence that contains the Kozak sequence and encodes the MERS-CoV consensus spike antigen ΔCD) Was included.

  The structure and properties of this MERS-HCoV-ΔCD construct are also described below in Examples 9 and 10.

Example 3
Expression and Humoral Response The MERS-HCoV-WT and MERS-HCoV-ΔCD constructs described above were examined to determine that the respective antigens are expressed intracellularly and can be recognized by antibodies. The MERS-HCoV-WT and MERS-HCoV-ΔCD constructs were transfected into 293T cells with pVAX1. pVAX1 served as a control.

  Cell lysates were prepared 2 days after transfection and run on a 5-15% SDS gel. Specifically, for cell lysates obtained from the 293T cells transfected with 10 μg, 25 μg, and 50 μg of cell lysate with either the MERS-HCoV-WT or MERS-HCoV-ΔCD construct, Loaded into wells. Next, immunoblot analysis was performed using serum from mice immunized with the MERS-HCoV-WT construct. No protein band was detected in cell lysates obtained from 293T cells transfected with pVAX1 (FIG. 9, pVAX1 labeled lane). In both the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs, protein bands corresponding to the expected molecular weight of the respective antigens were detected and from the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs. The expression of each antigen was confirmed (FIG. 9).

  The immunoblot showed that sera from mice immunized with the MERS-HCoV-WT construct recognized both the MERS-CoV consensus spike antigen and the MERS-CoV consensus spike antigen ΔCD, and It also showed that no other proteins were recognized (as evidenced by no band observed in cell lysates from 293T cells transfected with pVAX1). This serum was therefore specific for the consensus spike antigen. The immunoblot further showed that the MERS-HCoV-WT construct was immunogenic and produced a strong humoral response.

Example 4
Immunization Schedule of Examples 5-7 C57 / BL6 mice were immunized with pVAX1, MERS-HCoV-WT construct, or MERS-HCoV-ΔCD according to the immunization regime shown in FIG. On day 0, each mouse received its respective vaccine by intramuscular injection (IM) followed by electroporation. Specifically, each mouse was anesthetized with tribromoethanol-avatin, the hair of the anterior tibial muscle (TA) was shaved with a small animal clipper, and the skin was exposed. The vaccine was administered in a final volume of 30-50 μL by IM injection. A sterile CELLECTRA3P ID array was then inserted from the skin into the muscle surrounding the IM injection site. The CELLECTRA 3P ID array was 3 mm long and contained three 26 gauge needle electrodes joined together with molded plastic. The needle electrode was attached to the CELLECTRA electroporation apparatus by applying CELLECTRA3P. Following insertion of the array into the muscle, short electrical pulses were delivered and the immunized mice were placed in their respective cages and carefully observed until resuscitation. The immunization procedure was repeated on days 14 and 28. Thus, immunization on day 0 was a stimulation immunization, and immunizations on days 14 and 28 were additional immunizations. On day 35, the mice were sacrificed and immunoassays were performed as described in Examples 5-7 below.

Example 5
IgG antibody response To further investigate the humoral immune response induced by the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs, mice were immunized as described in Example 4. Blood was drawn prior to the start of the immunization regimen, and blood was also collected on days 21 and 35 of the immunization regimen. The pre-collection and blood collection were obtained by the retroorbital venous plexus method.

  Pre-bleed, 21-day, and 35-day blood draws from mice immunized with the MERS-HCoV-WT construct were examined and the concentration of IgG antibody immunoreactive with the peptide derived from the total consensus spike antigen Was measured. Specifically, the pre-bleeds and sera from each draw were diluted 1:50 and added to ELISA plates coated with the mixed peptide pool. The mixed peptide pool contained peptides from different parts of the total consensus spike antigen. Specifically, the mixed peptide pool was an equal mixture of the six linear peptide pools 1 to 6 described in Example 6 and FIG.

  As shown in FIG. 11A, the MERS-HCoV-WT construct elicited high concentrations of IgG antibodies that were immunoreactive with peptides derived from the entire consensus spike antigen. The data in FIG. 11A is from the sera of 4 mice and is expressed as mean ± standard error (SEM). These data indicate that the MERS-HCoV-WT construct (in comparison to the pre-bleed and blood draw) increased the concentration of IgG antibody that recognized the peptide from the total consensus spike antigen by about 4-fold to about 6-fold. Showed that it was induced.

  Pre-bleeds from mice immunized with the MERS-HCoV-ΔCD construct, blood draw on day 21, and blood draw on day 35 were also examined, and the concentration of IgG antibody immunoreactive with the peptide derived from the total consensus spike antigen Was measured. The pre-bleeds and serum from each draw were diluted 1:50 and added to ELISA plates coated with mixed peptide pools. The mixed peptide pool contained peptides from different parts of the total consensus spike antigen. Specifically, the mixed peptide pool was an equal mixture of the six linear peptide pools 1 to 6 described in Example 6 and FIG.

  As shown in FIG. 11B, the MERS-HCoV-ΔCD construct elicited high concentrations of IgG antibodies immunoreactive with the peptides derived from the whole consensus spike antigen. The data in FIG. 11B is from the sera of 4 mice and is expressed as mean ± SEM. These data indicate that the MERS-HCoV-ΔCD construct (in comparison to the pre-bleed and blood draw) increased the concentration of IgG antibody that recognized the peptide from the total consensus spike antigen by about 6 to 8 times. Showed that it was induced.

  In summary, the data in FIGS. 11A and 11B show that both the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs induced high concentrations of IgG antibodies immunoreactive with peptides derived from the whole consensus spike antigen. Showed that. These data also indicated that the MERS-HCoV-ΔCD construct elicited a higher concentration of immunoreactive IgG antibody than the MERS-HCoV-WT construct. Therefore, the MERS-HCoV-ΔCD construct was more immunogenic than the MERS-HCoV-WT construct as judged by IgG antibody response.

Example 6
CD8 ⁺ T cell response As noted above, the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs each induced a significant humoral immune response against the MERS-CoV spike antigen. To ascertain whether the MERS-HCoV-WT and MERS-CoV-ΔCD constructs also induce a cellular immune response, mice were immunized as described in Example 4. Coverage of the amino acid sequence of the entire consensus MERS-CoV spike antigen using the interferon-gamma (IFN-γ) ELISpot assay for antigen-specific responses of CD8 ⁺ T cells after 35 days of killing of the immunization regimen The peptide pool matrix was analyzed. The peptide pool matrix is shown in FIG. 12 and facilitated the identification of dominant epitopes recognized by the CD8 ⁺ T cell response induced with the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs. The matrix for this peptide pool was obtained from a peptide scan over the full length of the MERS-HCoV-WT spike antigen, which was a 15-mer overlapping 11 amino acids.

The results of IFN-γ ELISpot analysis on this peptide pool matrix are shown in FIGS. 13 and 14 for the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs, respectively. In both FIGS. 13 and 14, data were expressed as mean ± SEM. Both constructs induced a significant CD8 ⁺ T cell response and dominant epitopes were present in pools 18, 19, 20, and 21 (FIGS. 13 and 14, circled bars). Another epitope was present in pool 4. Therefore, these data indicated that the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs induced a strong cellular immune response reactive with a subset of spike antigen peptides.

To further investigate the CD8 ⁺ T cell response, mice were immunized with 25 μg of pVAX1, MERS-HCoV-WT construct, or MERS-HCoV-ΔCD construct according to the immunization regime described in Example 4 above. Immunization with pVAX1 served as a control. Following the 35 day kill of the immunization regimen, the antigen-specific response of CD8 ⁺ T cells isolated from 3 groups of mice was examined using IFN-γ ELISpot analysis. Specifically, the CD8 ⁺ T cells were stimulated with 6 different peptide pools. These peptide pools 1-6 covered the peptides shown in the matrix of FIG.

As shown in FIG. 15, the magnitude of the cellular immune response induced with the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs stimulates CD8 ⁺ T cells isolated from each mouse. It varied depending on the peptide pool used. For both the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs, peptide pool 1 elicits a CD8 ⁺ T cell response similar to control pVAX1, thereby stimulating CD8 ⁺ T cells. It indicated that it did not contain an epitope. Peptide pools 1-6 elicited similar responses from the CD8 ⁺ T cells isolated from mice immunized with pVAX1.

Peptide pools 2, 3, and 4 elicited comparable responses from CD8 ⁺ T cells isolated from mice vaccinated with MERS-HCoV-WT or MERS-HCoV-ΔCD constructs. Furthermore, the CD8 ⁺ T cell response to peptide pools 2, 3, and 4 is compared to the pVAX1 control when mice are immunized with either the MERS-HCoV-WT or MERS-HCoV-ΔCD construct. Significantly higher (FIG. 15). Specifically, the CD8 ⁺ T cell response to peptide pools 2, 3, and 4 was about 7 times higher when mice were immunized with the MERS-HCoV-ΔCD construct compared to control pVAX1. . The CD8 ⁺ T cell responses to peptide pools 2, 3, and 4 were about 7.5 times and about 9 times higher when mice were immunized with the MERS-HCoV-WT construct, respectively, compared to the control pVAX1. Doubled and about 10 times higher. Thus, these data indicate that the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs differed from the control pVAX1 in significant CD8 ⁺ T cell responses to peptides contained within peptide pools 2, 3, and 4. It was shown that it was induced.

Peptide pools 5 and 6 were compared to peptide pools 1, 2, 3, and 4 with the CD8 ⁺ T isolated from mice immunized with the MERS-HCoV-WT or MERS-HCoV-ΔCD construct. A higher response was elicited from the cells (FIG. 15). Furthermore, the CD8 ⁺ T cell response to peptide pools 5 and 6 was significant compared to control pVAX1 when mice were immunized with either the MERS-HCoV-WT or MERS-HCoV-ΔCD construct. It was expensive. Specifically, CD8 ⁺ T cell responses to peptide pools 5 and 6 were about 9 and 11 times higher, respectively, when mice were immunized with the MERS-HCoV-ΔCD construct compared to control pVAX1. . CD8 ⁺ T cell responses to peptide pools 5 and 6 were about 13-fold and about 15-fold higher when mice were immunized with the MERS-HCoV-WT construct, respectively, compared to control pVAX1. Thus, these data indicate that the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs induced a significant CD8 + T cell response to peptides contained in peptide pools 5 and 6, unlike the control pVAX1. Indicated.

  In summary, the data show that immunization with the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs induced significant cellular immune responses that were specific and reactive with a subset of spike antigen peptides. It was.

Example 7
Multifunctional Cellular Immune Response As mentioned above, the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs induced a CD8 ⁺ T cell response that was reactive to the MERS-CoV spike antigen. To further investigate the cellular immune response induced with the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs, the functionality of the T cells after immunization was examined. Specifically, mice were immunized with pVAX1, MERS-HCoV-WT construct, or MERS-HCoV-ΔCD construct according to the immunization regime described in Example 4 above. Splenocytes were isolated from sacrificed mice on day 35 of the immunization regimen. Isolated splenocytes were stimulated in vitro with a peptide pool containing peptides from the whole consensus MERS-CoV spike antigen. The peptide pool and the splenocytes were cultured together for 5 hours. Cells were then stained for intracellular production of IFN-γ, tumor necrosis factor alpha (TNF-α), and interleukin-2 (IL-2) and sorted with a fluorescence activated cell sorter (FACS).

16A, 16B, 16C, and 16D show stimulated splenocyte populations of IFN-γ, TNF-α, IL-2, or CD3 ⁺ CD4 ⁺ T cells that produce both IFN-γ and TNF-α, respectively. The frequency measured with is shown. The data in FIGS. 16A-16D represented splenocytes isolated from 3 mice for each group. The data of FIGS. 16A-16D were also expressed as mean ± SEM. The frequency of IFN-γ, TNF-α, IL-2, or CD3 ⁺ CD4 ⁺ T cells producing both IFN-γ and TNF-α was the control in the MERS-HCoV-WT and MERS-HCoV-ΔCD groups Significantly increased compared to the pVAX1 group.

Specifically, the frequency of CD3 ⁺ CD4 ⁺ IFN-γ ⁺ T cells was compared to the control pVAX1 in splenocyte populations isolated from mice immunized with the MERS-HCoV-ΔCD and MERS-HCoV-WT constructs. In comparison, they were about 6 times and about 13 times larger (FIG. 16A). The frequency of CD3 ⁺ CD4 ⁺ TNF-α ⁺ T cells is about 30% compared to the control pVAX1, respectively, in splenocyte populations isolated from mice immunized with the MERS-HCoV-ΔCD and MERS-HCoV-WT constructs. It was 6 and about 11 times larger (FIG. 16B). The frequency of CD3 ⁺ CD4 ⁺ IL-2 ⁺ T cells is approximately about each compared to control pVAX1 in splenocyte populations isolated from mice immunized with the MERS-HCoV-ΔCD and MERS-HCoV-WT constructs. It was 12.5 times larger and about 30 times larger (FIG. 16C). CD3 ⁺ CD4 ⁺ IFN-γ ⁺ TNF-α ⁺ T cell frequency compared to control pVAX1 in splenocyte populations isolated from mice immunized with the MERS-HCoV-ΔCD and MERS-HCoV-WT constructs And about 7.5 times and about 17.5 times larger, respectively (FIG. 16D). Thus, these data indicate that the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs, unlike the control pVAX1, induced a multifunctional T cell response and increased numbers of CD3 + CD4 + T cells were IFN-γ, TNF- It was shown that α, IL-2, or both IFN-γ and TNF-α were produced.

Figures 17A, 17B, 17C, and 17D show stimulated splenocyte populations of IFN-γ, TNF-α, IL-2, or CD3 ⁺ CD8 ⁺ T cells that produce both IFN-γ and TNF-α, respectively. The frequency measured with is shown. The data in FIGS. 17A-17D represented splenocytes isolated from 4 mice for each group. The data in FIGS. 17A-17D were also expressed as mean ± SEM. The frequency of CD3 ⁺ CD8 ⁺ T cells producing IFN-γ, TNF-α, IL-2, or both IFN-γ and TNF-α is the MERS-HCoV-WT and MERS-HCoV-ΔCD groups, Significantly increased compared to control pVAX1.

Specifically, the frequency of CD3 ⁺ CD8 ⁺ IFN-γ ⁺ T cells was compared to control pVAX1 in splenocyte populations isolated from mice immunized with the MERS-HCoV-ΔCD and MERS-HCoV-WT constructs. In comparison, they were about 8 times and about 13 times larger (FIG. 17A). The frequency of CD3 ⁺ CD8 ⁺ TNF-α ⁺ T cells was approximately 7 compared to control pVAX1 in splenocyte populations isolated from mice immunized with the MERS-HCoV-ΔCD and MERS-HCoV-WT constructs. It was twice as large (FIG. 17B). The frequency of CD3 ⁺ CD8 ⁺ IL-2 ⁺ T cells is approximately 2 compared to control pVAX1 in splenocyte populations isolated from mice immunized with the MERS-HCoV-ΔCD and MERS-HCoV-WT constructs. .5 times larger (FIG. 17C). CD3 ⁺ CD8 ⁺ IFN-γ ⁺ TNF-α ⁺ T cell frequency compared to control pVAX1 in splenocyte populations isolated from mice immunized with the MERS-HCoV-ΔCD and MERS-HCoV-WT constructs And about 50 times and about 90 times larger, respectively (FIG. 17D). Thus, these data indicate that the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs, unlike the control pVAX1, induced a multifunctional T cell response, and an increased number of CD3 ⁺ CD8 ⁺ T cells were IFN- It was shown that γ, TNF-α, IL-2, or both IFN-γ and TNF-α were produced.

In summary, the above data indicate that the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs produced IFN-γ, TNF-α, IL-2, or both IFN-γ and TNF-α. It was shown that CD3 ⁺ CD4 ⁺ and CD3 ⁺ CD8 ⁺ T cells were significantly induced. The MERS-HCoV-WT and MERS-HCoV-ΔCD constructs supported more multifunctionality of both CD3 ⁺ CD4 ⁺ and CD3 ⁺ CD8 ⁺ T cells than the control pVAX1. Thus, the consensus construct was able to elicit a multifunctional cellular immune response reactive with the MERS-CoV spike antigen.

Example 8
Neutralizing antibody As described in Example 5 above, the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs induced a humoral immune response. To further investigate the induced humoral immune response, neutralizing antibody concentrations of mice immunized with pVAX1, MERS-HCoV-WT construct, or MERS-HCoV-ΔCD construct were examined. Specifically, the serum was diluted with the minimum necessary amount of medium and cultured at 37 ° C. with 50 μl of DMEM containing 100 infectious HCoV-EMC / 2012 (Human Coronaviras Erasmus Medical Center / 2012) per well. After 90 minutes, the virus-serum mixture was added to Vero cell monolayers (100,000 cells / well) in 96-well flat bottom plates and cultured at 37 ° C. in a 5% CO ₂ incubator for 5 days. The neutralizing antibody titer for each specimen was shown as the maximum dilution at which less than 50% of the cells showed CPE. Values are given as reciprocal dilutions. All the samples were duplicated and the final result was the average of the two samples.

  These results are shown in Table 1. Immunization with the MERS-HCoV-WT and MERS-HCoV-ΔCD constructs induced significant concentrations of neutralizing antibodies reactive with the MERS-CoV spike antigen, unlike the control pVAX1.

In summary, the data provided in these examples herein show that the MERS-HCoV-WT and MERS-CoV-ΔCD constructs are effective vaccines and are humoral in reactivity with the MERS-CoV spike antigen. And proved to significantly induce both cellular immune responses. The induced humoral immune response includes increased titers of IgG and neutralizing antibodies immunoreactive with the MERS-CoV spike antigen compared to a construct lacking either consensus antigen (ie, pVAX1) It is. The induced cellular immune response includes increased IFN-γ, TNF-α, IL-2, or IFN-γ and TNF-α compared to the construct lacking either consensus antigen (ie, pVAX1). Includes responses of both CD3 ⁺ CD4 ⁺ and CD3 ⁺ CD8 ⁺ T cells that produced both.

Example 9
Materials and Methods of Examples 10-19 Cell Culture, Plasmid, and Expression of MERS-HCoV-Spike Protein HEK293T cells (ATCC #: CRL-N268) and Vero-E6 cells (ATCC #: CRL-1586) are 10% FBS Growing in DMEM (DMEM). The MERS-HCoV-Spike WT and SpikeΔCD plasmid DNA constructs encoded an optimized consensus sequence for the MERS Spike protein (S). In addition, Ig heavy chain epsilon-1 signal peptide was linked to the N-terminus of each sequence and replaced with N-terminal methionine to promote expression. Each gene was genetically optimized for expression in mice, including codon optimization and RNA optimization. The optimized gene was then subcloned into a modified pVax1 mammalian expression vector under the control of the cytomegalovirus immediate early (CMV) promoter (GenScript). The structures of these MERS-HCoV-Spike WT and SpikeΔCD plasmid DNA constructs are also described in Examples 1 and 2 above.

  For in vitro expression studies, transfection was performed using TurboFectin 8.0 reagent according to the manufacturer's protocol (OriGene). Briefly, cells were grown to 80% confluence in 35 mm dishes and transfected with 3 μg Spike plasmid. The cells were harvested 48 hours (h) after transfection, washed twice with phosphate buffered saline (PBS), then suspended in cell lysis buffer (Cell Signaling Technology) and spike protein by Western blot analysis. The expression of was verified.

Mice and Immunization Female C57BL-6 mice (6-8 weeks old; Jackson Laboratories) were used for these experiments and divided into three experimental groups. All animals were maintained at the National Institutes of Health (Beethsda) and University of Pennsylvania (Philadelphia, Pa., USA) at the Institutional Animal Care and Use Committee (IACUC), maintained by the Institute of Light and Cycle Management, IACUC guidelines. All immunizations were administered to the anterior tibial muscle (25 μg) using a minimally invasive EP supply technique (MID-EP) in vivo.

Single cell suspensions of spleen were obtained from the immunized mice. That is, spleens of mice immediately after euthanasia were individually collected in 10 ml of RPMI 1640 (R10) supplemented with 10% FBS, and then paddle blender (STOMACHER 80 paddle blender; AJ Seward and Co. Ltd., London, UK). ) At a high speed for 60 seconds. The treated spleen specimen was filtered through a 45 μm nylon filter, and then centrifuged at 800 × g for 10 minutes at room temperature. The cell pellet was resuspended in 5 ml of ACK lysis buffer (Life Technology) for 5 minutes at room temperature, and then PBS was added to stop the reaction. The specimen was again centrifuged at 800 × g for 10 minutes at room temperature. The cell pellet was suspended in R10 at a concentration of 1 × 10 ⁷ cells / ml and then passed through a 45 μm nylon filter prior to use for enzyme-linked immunosorbent spot (ELISpot) analysis and flow cytometry analysis.

ELISpot analysis Antigen-specific T cell responses were measured using IFN-γ ELISpot. Specifically, PVDF 96-well plates (Millipore) were coated with purified anti-mouse IFN-γ capture antibody and cultured at 4 ° C. for 24 hours (R & D Systems). The next day, the plates were washed and blocked with 1% BSA and 5% sucrose for 2 hours. 200,000 splenocytes from the immunized mice are added to each well at 37 ° C. in 5% CO ₂ , RPMI 1640 (negative control), ConA (5 ug / mL; positive control), or specific peptide antigen ( Ag) (10 μg / ml; GenScript) was stimulated overnight. The peptide pool consisted of 15-mer peptides overlapping 11 amino acids (GenScript). Twenty-four hours after stimulation, the cells were washed and cultured with biotinylated anti-mouse IFN-γ Ab (R & D Systems) for 24 hours at 4 ° C. The plate was washed and streptavidin alkaline phosphatase (R & D Systems) was added to each well and incubated for 2 hours at room temperature. The plate was washed and 5-bromo-4-chloro-3'-indolyl phosphate p-toluidine salt and nitroblue tetrazolium chloride (Chromogen color reagent; R & D Systems) were added to each well. The plate was then rinsed with distilled water and allowed to dry overnight at room temperature. Spots were counted with an automatic ELISpot reader (CTL Limited).

Flow Cytometry and Intracellular Cytokine Staining (ICCS) Analysis Splenocytes were added to 96-well plates (1 × 10 ⁶ / well) and pooled MERS antigen peptide with Protein Transport Inhibitor Cocktail (Brefeldin A and Monensin; eBioscience) Was stimulated with 37 C / 5% CO ₂ for 5-6 hours according to the manufacturer's instructions. Cell Stimulation Cocktail (further protein transport inhibitor) (phorbol 12-myristic acid 13-acetate (PMA), ionomycin, brefeldin A and monensin; eBioscience) was used as a positive control and R10 medium was used as a negative control. All cells were then stained for surface and intracellular proteins as per manufacturer's instructions (BD). That is, the cells were washed with FACS buffer (PBS containing 0.1% sodium azide and 1% FCS) before surface staining with a fluorescent dye-labeled antibody. Cells were washed with FACS buffer, fixed, permeabilized using BD Cytofix / Cytoperm ™ (BD) according to the manufacturer's protocol, followed by intracellular staining. The following antibodies were used for surface staining: LIVE / DEAD Fixable Violet Dead Cell staining kit (Invitrogen), CD19 (V450; clone 1D3; BD Biosciences) CD4 (FITC; clone RM4-5; ebioscience), CD8 (APC-Cy7) Clone 53-6.7; BD Biosciences); CD44 (A700; clone IM7; Biolegend). For intracellular staining, the following antibodies were used: IFN-γ (APC; clone XMG1.2; Biolegend), TNF-α (PE; clone MP6-XT22; bioscience), CD3 (PerCP / Cy5.5; clone) 145-2C11; Biolegend); IL-2 (PeCy7; clone JES6-SH4; ebioscience). All data was collected using an LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star) and SPICE v5. Bourgating was performed using FlowJo software to examine the polyfunctionality of T cells from vaccinated animals.

Immunogen specific ELISA
Mouse serum titers were measured using enzyme-linked immunosorbent assay (ELISA). That is, using a purified recombinant human beta coronavirus Spike protein 2c EMC / 2012 (branch group A) (Sino Biological Inc.) 5 μg / ml, a 96-well microtiter plate (Nalgene Nunc International, Naperville, Ill.) At 4 ° C. And coated overnight. After blocking with FBS 10% PBS, the plates were washed 4 times with 0.05% PBST (PBS with Tween 20). Serum specimens of immunized mice were serially diluted with 1% FBS and 0.2% PBST, added to the plate, and then incubated at room temperature for 1 hour. The plates were again washed 4 times with 0.05% PBST and incubated for 1 hour at room temperature with HRP-labeled anti-mouse IgG diluted according to the manufacturer's instructions (Sigma Aldrich). Bound enzyme was detected by adding OPD (o-phenylenediamine dihydrochloride) tablets according to the manufacturer's instructions (SIGMAFAST; Sigma-Aldrich). The reaction was stopped after 15 minutes by addition of 2N H ₂ SO ₄ . The plate was then read at 450 nm optical density on a 96 Microplate Luminometer (GLOMAX; Promega). All specimens were repeated into plates. The endpoint titer was determined as the maximum dilution of the mean absorbance from 3 or more normal sera plus an absorbance value higher than 3 standard deviations.

Indirect immunofluorescence analysis (IFA)
For cells transfected with MERS-HCoV-Spike plasmid, glass slides were fixed with ice-cold acetone for 5 minutes. Nonspecific binding was then blocked with PBS containing 5% skim milk for 30 minutes at 37 ° C. The slides were then placed in PBS and washed for 5 minutes, followed by incubation with immunized mouse serum at a 1: 100 dilution. After incubation for 1 hour, the slides were washed as described above, with Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) diluted in PBS containing 1 ug / mL of 4 6-diamidino-2-phenylindole (DAPI) at 37 ° C. Incubated for 30 minutes. After washing away unbound antibody, Prolong Gold antifade reagent (Invitrogen) was placed on the coverslip and the glass slide was observed under a confocal microscope (LSM710; Carl Zeiss). The obtained image was analyzed using Zen software (Carl Zeiss).

Production of MERS-HCoV-Spike pseudovirus particles The codon optimized consensus MERS-HCoV Spike gene was synthesized and subcloned into the pVax1 vector. A MERS-HCoV-Spike pseudovirus was obtained. Specifically, 2 × 10 ⁶ HEK293T cells are seeded in a 10 cm tissue culture plate, and the MERS-Spike or VSV-G protein is about 80% confluent with TurboFectin 8.0 (OriGene) transfection reagent. Transfected with the expressing plasmid. To produce HIV / MERS-Spike pseudoparticles, cells were co-transfected with 10 μg pNL Luc ER- (NIH-AIDS-Reagent Program) and 10 μg pVax1-MERS-Spike from various branch groups. After 12 hours, the transfection medium was removed, replaced with fresh medium for about 12 hours, and the cells were cultured at 37 ° C. for 24-48 hours. The pseudovirus-containing medium was collected and filtered, and the pseudovirus was passed through a 20% sucrose cushion prepared with TNE buffer (Tris 10 mM, NaCl 135 mM, EDTA 2 mM, pH 8.0) for 1 hour at 40,000 rpm in a SW41 rotor. And concentrated. The pellet was resuspended in 500 μL of TNE buffer overnight, divided and stored at −80 ° C.

Calculated neutralized analyzed 50% tissue culture infectious dose of (TCID _50), the standard concentration of virus (i.e., 100 TCID ₅₀₎ and the neutralization test throughout the studies conducted in mice sera from MERS-HCoV DNA immunized animals Using. That is, the mouse serum was serially diluted with MEM and cultured at 37 ° C. with 50 ul of DMEM containing 100 infectious HCoV-EMC / 2012 (Human Coronavirus Erasmus Medical Center / 2012) per well. After 90 minutes, the virus-serum mixture was added to Vero cell monolayers (100,000 cells / well) in 96-well flat bottom plates and cultured at 37 ° C. in a 5% CO ₂ incubator for 5 days. The neutralizing antibody titer for each specimen was expressed as the maximum dilution at which less than 50% of the cells showed CPE. Values are given as reciprocal dilutions. All samples were tested in duplicate and the final result was the average of the two values. The neutralization rate was calculated as follows: Neutralization rate = {1-PFU of target mAb (each concentration) / average PFU of negative control (total concentration)}. A neutralization curve was created and analyzed using GraphPad Prism 5. IC ₅₀ and IC ₈₀ were measured using non-linear regression fitting with sigmoidal dose-response (variable slope). Positive and negative control sera were included to validate this analysis.

  For the neutralization test, MERS-Spike pseudoparticles (50 ng) were preincubated with serially diluted mouse serum for 30 minutes at 4 ° C. and then added to the cells in triplicate. Three days after infection, the CPE was read. The maximum serum dilution that completely protected the cells from CPE in half of the wells was taken as the neutralizing antibody titer. For luciferase-based analysis, cells infected with MERS-Spike pseudoparticles were lysed 2 days after infection with 50 μl protein lysis buffer and 100 μl luciferase substrate. Luciferase activity was measured with 96 Microplate Luminometer (GLOMAX; Promega Corporation).

Cell viability analysis and Annexin V / FITC analysis Cell viability was measured using 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT, 5 mg / ml, Sigma) . Culture was initiated in 96-well plates with a cell density of 2.5 × 10 ³ per well. After 48 hours of culture, the cells were infected with MERS pseudovirus and cultured for 48 hours. After incubation, 15 μl of MTT reagent was added to each well and incubated at 37 ° C. for 4 hours in the dark. The supernatant was aspirated and the formazan crystals were dissolved in 100 μl of DMSO with gentle stirring at 37 ° C. for 15 minutes. Absorbance at 540 nm per well was measured using a Luminometer Reader (GLOMAX; Programmer). Data were analyzed from three independent experiments and normalized to the absorbance of wells containing medium only (0%) and untreated cells (100%). IC ₅₀ values were calculated from sigmoidal dose-response curves using Prism GraphPad software.

Annexin V / FITC binding analysis was performed using Annexin V-FITC detection kit (BD Biosciences) according to the manufacturer's instructions. The cells were infected with MERS pseudovirus for 12 hours. The cells were counted after trypsinization and washing twice with cold PBS. The cell pellet was resuspended in 100 μl binding buffer at a cell density of 1 × 10 ³ per ml and incubated for 15 minutes at room temperature in the dark with 5 μl FITC-labeled Annexin-V and 5 μl PI. 400 μl of 1 × binding buffer was added to each sample tube and the sample was immediately analyzed with a flow cytometer (BD Biosciences). The data was analyzed using FlowJo software (Tree Star).

Non-human primate (NHP) immunization and MERS challenge Three groups of rhesus monkeys were administered three times (stimulation and two additional), three weeks apart (weeks 0, 3, 6). Group 1 and Group 2 rhesus monkeys received a total of 0.5 mg and 2 mg / dose of DNA vaccine expressing the MERS-Spike (N = 4) vaccine by intramuscular immunization with an EP device. . Groups 1 and 2 received the entire consensus MERS Spike antigen. Group 3 rhesus monkeys received a total of 2 mg / dose of the control vaccine (pVax1). The dosage and immunization regimen of DNA vaccines in these studies were previously determined to be optimal in rhesus monkeys. Blood was collected after each administration to analyze serum antibodies and neutralization and systemic T cell responses. The animals were anesthetized with an intramuscular injection of ketamine HCL (10-30 mg / kg). The vaccine was administered to each thigh (one injection site per thigh for each inoculation) and was delivered by the intramuscular (IM) route. For IM administration, immediately after DNA injection, 3 pulses with a constant current of 0.5 A and a duration of 52 ms were applied at a pulse interval of 1 s.

NHP challenge Three groups of rhesus monkeys were dosed 3 times (stimulation and 2 additions), 3 weeks apart (0, 3 and 6 weeks). Group 1 and Group 2 rhesus monkeys received DNA vaccines expressing MERS-Spike (N = 4) vaccine in a total of 0.5 mg and 2 mg / dose by intramuscular immunization with an EP device. Group 3 rhesus monkeys received a total of 2 mg / dose of the control vaccine (pVax1). The dosage and immunization regimen of DNA vaccines in these studies were previously determined to be optimal in rhesus monkeys. Blood was collected after each administration to analyze serum antibodies and neutralization and systemic T cell responses. The animals were anesthetized with an intramuscular injection of ketamine HCL (10-30 mg / kg). The vaccine was administered to each thigh (one injection site per thigh for each inoculation) and was delivered by the intramuscular (IM) route. For IM administration, immediately after DNA injection, 3 pulses with a constant current of 0.5 A and a duration of 52 ms were applied at a pulse interval of 1 s.

Challenge Twelve healthy rhesus monkeys (Macara mulatta) aged 4-6 years, as already established (Falzarano et al Nature Medicine 19, 1313-1317 (2013)) intratracheal, intranasal, oral, and ocular A total of 7 × 10 ⁶ TCID50 was inoculated with MERS-CoV. Animals were randomly assigned to treated or untreated groups in an open-label manner.

Statistical analysis The animal experiment to assess immune response was repeated at least three times and the response of each mouse was taken as an individual data point for statistical analysis. All data are expressed as mean ± standard deviation. Data from animal experiments and various immunoassays were examined using GraphPad's one-way ANOVA, and the difference was considered significant at p <0.05. GraphPad Prism v. 5.0 (GraphPad Software, Inc.) was used for statistical analysis.

Example 10
Cloning and expression of the MERS-HCoV Spike and SpikeΔCD proteins The consensus sequence for the MERS-HCoV Spike gene was generated from 16 Spike genomic sequences in the GenBank-NCBI database. For immunogen design, include sequences from both branch groups A and B in the consensus sequence and measure the resulting consensus sequence for neutralizing activity across MERS-HCoV branch groups A and B virus strains. Analyzed with As shown in FIG. 19A, the systematic position of the MERS-HCoV-Spike consensus sequence was centered in the branch group B quadrant because many sequences are included in this branch group. In addition, two consensus sequences for the MERS Spike glycoprotein were designed where the cytoplasmic domain sequence of one construct remained intact and the cytoplasmic domain of the second construct was excised. These constructs were designated MERS-HCoV Spike (Spike-Wt) and partial cytoplasmic defect (SpikeΔCD), respectively. For both immunogens, several modifications were made to improve in vivo expression, including the addition of a highly efficient IgE leader peptide sequence to promote mRNA nuclear export. The insert was subcloned into the pVax1 vector (FIG. 19B). The structures of these Spike-Wt and SpikeΔCD DNA constructs are also described in Examples 1 and 2 above.

  These plasmids were separately transfected into 293T cells and Spike protein expression was assessed by Western blot. Mouse serum specific for Spike-Wt protein from the immunized mice was used to detect Spike protein expression in cell lysates transfected with the plasmid. Forty-eight hours after transfection, protein lysates were extracted. A strong specific band of MERS-Spike protein (140 kDa) was detected in Spike-Wt and SpikeΔCD transfected cells, but not in lysates from cells transfected with the control vector (empty pVax1 plasmid) (FIG. 19C). ).

  In addition, Spike protein expression and localization after transfection was examined using immunofluorescence analysis (IFA). With IFA using the mouse Spike antiserum, it was found that a strong signal was present in the cytoplasm (FIG. 19D). A positive signal was not detected in intact cells transfected with the pVax1 vector. Both the entire construct (Spike-Wt) and the cytoplasmic domain mutant construct (SpikeΔCD) were similarly localized to the transfected cells. These results demonstrate the strong ability of the MERS-HCoV constructs to be expressed in mammalian cells and that the antibodies derived from these constructs can bind to their target antigens.

Example 11
Functional expression and infection with MERS-HCoV DNA constructs To measure the functionality (eg, virus binding and entry) of the consensus sequence immunogen, a pseudoviral expression system was developed to analyze the virus entry of the consensus construct . Specifically, MERS-HCoV pseudoviral particles were prepared by co-transfecting 293T cells with a plasmid encoding the MERS-Spike antigen and an HIV-1 luciferase reporter plasmid that does not express the HIV-1 envelope (Figure 20A). Particles were produced by transfecting 293T cells with various MERS-Spike genes + lentiviral genomic fragments. This causes robust virus particle formation. To characterize MERS-Spike mediated infection, luciferase activity of cells infected in synchrony with the consensus MERS-Spike pseudovirus was measured and analyzed over time (FIG. 20B). As seen in FIG. 20C, inoculation of Vero and A549 cell lines expressing a functional receptor for MERS-CoV with pseudoviral particles produced a strong luciferase signal, indicating a productive infection with the pseudovirus. It was. These experiments demonstrated the development of a MERS pseudovirus with Spike protein that entered target cells and established a pseudovirus-based inhibition analysis for detection of neutralizing antibodies.

Example 12
Increased immunogenicity of MERS-HCoV DNA vaccine in mice To examine the immunogenicity of the consensus and modified Spike glycoprotein, constructs were analyzed for their ability to elicit immune responses after intramuscular injection in C57BL / 6 mice. Female C57BL / 6 mice (n = 9) were vaccinated with 25 μg of one of three DNA plasmids: Spike-Wt, SpikeΔCD, or control pVax1 vector. Immediately after each immunization, an adaptive electroporation (EP) system was used. As shown in FIG. 21A, animals were vaccinated 3 times at 2 week intervals and the immune response was measured one week after the third immunization.

  Cell-mediated immunity was assessed using standard ELISpot analysis, and cytokines following antigen-specific in vitro restimulation of splenocytes from the immunized mice with a peptide pool encoding the entire protein region of the MERS-Spike glycoprotein Secretory capacity was monitored. ELISpot analysis was performed one week after the third immunization. As shown in FIG. 21B, splenocytes from Spike-Wt and immunized SpikeΔCD vaccination induced strong cellular immune responses against multiple peptide pools. The peptides in pools 2 and 5 appeared dominant in both constructs.

Based on these T cell responses, detailed mapping was performed for 31 different pools spanning the entire MERS-Spike protein. A 227 peptide pool was created, containing 15-mer peptides overlapping 11 amino acids and spanning residues of Spike protein. 31 peptide pools were prepared and tested using matrix type. After restimulation with the peptide, a strong CD8 ⁺ T cell response was detected for several regions with the Spike protein (FIGS. 21C and 21D). There were 15 matrix pools showing over 100 spots, indicating that MERS-Spike elicited a wide range of cellular immune responses. Four peptide pools were identified in the region from amino acids 301-334. Importantly, the Spike-Wt and Spike-ΔCD immunogens responded to four major regions spanning peptide pools 4-6, 11-13, 18-21, and 29-31. However, the dominant pool appeared to range from 18-21. These pools contained a computer-identified CD8 ⁺ T lymphocyte immunodominant epitope at amino acids 307-321 (RKAWAAFYVYKLQPL (SEQ ID NO: 7)) and could be a dominant response by both antigens (FIGS. 21C and 21D).

Example 13
The T cell response induced with the MERS-Spike vaccine was multifunctional To further establish the phenotype of the induced T cell response, the multifunctional T cell response was evaluated. Polychromatic flow cytometry was used to measure the production of IFN-γ, IL-2, and TNF-α induced in an antigen-specific manner in both CD4 ⁺ and CD8 ⁺ T cells. Representative flow cytometry profiles of MERS-Spike specific IL-2, TNF-α, and IFN-γ secreting CD4 ⁺ and CD8 ⁺ T cells are shown in FIGS. 25A and 25B, respectively. The construct produced an equivalent response from CD8 ⁺ T cells, and the full length construct induced a significantly higher proportion of CD4 ⁺ T cells secreting IL-2, TNF-α, and IFN-γ.

The magnitude of vaccine induced CD4 ⁺ and CD8 ⁺ T cell responses were compared for the MERS-HCoV-Spike and MERS-HCoVΔCD vaccine constructs. Bourgating was used to evaluate the ability of individual cells to produce multiple cytokines, ie, multifunctionality of vaccine induced CD4 ⁺ and CD8 ⁺ T cell responses (FIGS. 25C and 25D). This analysis showed that the CD8 ⁺ T cell response was the same in the full length or defective Spike protein vaccine group, but the magnitude of the CD4 ⁺ T cell response was greater in the full length Spike antigen vaccine group. Seven different Spike specific CD4 ⁺ and CD8 ⁺ T cell populations were identified. Although the proportion of tri-, bi-, and monofunctional cells was slightly different in these two vaccine groups, in both groups, the overall magnitude of CD4 ⁺ and CD8 ⁺ T cell response induction both favored full length constructs A trend was observed. The response is then further divided into a combination of these seven possible functions, with the exception of induction of CD8 ⁺ T cells that produce IFN-γ that favors the full-length construct in the magnitude of the response as well. It was observed that the CD8 ⁺ T cells in the vaccinated groups were similar (FIGS. 25C and 25D).

Example 14
Sufficient binding and neutralizing antibody (Nab) responses induced by MERS-HCoV-Spike vaccination in mice were also examined for humoral immunity induction by the two constructs. Serum samples were collected before and after DNA immunization in mice. Anti-Spike humoral immune responses were analyzed for binding to recombinant Spike antigen and in functional antibody studies. As shown in FIGS. 18A and 22A, all vaccinated animals elicited specific antibody responses compared to control animals immunized with the plasmid backbone. Similar to the CD4 ⁺ T cell response, bound antibody activity was quantified as endpoint titer and was stronger in full length immunized animals (FIGS. 18B and 22B). The antibody produced in the immunized mice also bound the MERS-Spike recombinant protein in Western blot analysis (FIG. 22C). Overall, these data indicated that the Spike DNA vaccine induced specific antibody production and antibodies that specifically bound to the target Spike antigen.

Example 15
Anti-MERS-HCoV-Spike serum demonstrated neutralizing activity against MERS virus The neutralizing activity of sera from mice immunized with either Spike-Wt or SpikeΔCD vaccines was compared to HCoV-, a branching group A strain virus. Evaluated by virus neutralization analysis with EMC / 2012 (Human Coronavirus Erasmus Medical Center / 2012). Serum was collected from mice vaccinated with either Spike-Wt or SpikeΔCD and negative control pVax1 (n = 9 / group). As shown in FIGS. 18C and 22D, both vaccines induced neutralizing antibody titers significantly higher than serum titers from mice immunized with the control vector (pVax1) alone (p = 0.018). Similar to the antibody binding assay, although not significantly different, the deletion construct appeared to induce a lower concentration of neutralization response than the full-length construct.

  Since neutralization analysis is limited due to the lack of currently available full-length viruses, the antisera's MERS-Spike pseudovirus neutralizing ability was analyzed using the pseudoparticle neutralization analysis described above. As shown in FIG. 22E, mice (n = 4) antiserum vaccinated with MERS-Spike-Wt neutralized Vero cell infection by MERS-Spike pseudovirus, 120-960 and above and 50% neutralization Abs titer effectively inhibited. Furthermore, the sera were evaluated for neutralizing activity against different branching groups of MERS-CoV infection using inhibition analysis based on MERS pseudovirus (see FIG. 24D). FIG. 24D shows detection of neutralizing antibodies in vaccinated sera against MERS-CoV infection. In other words, monkey serum 2 weeks after the third immunization was collected from the vaccinated animals, and inhibition analysis based on MERS-CoV pseudovirus in Vero cells was performed for the low and high dose groups of MERS. . Pseudovirus invasion was quantified by luciferase activity 40 hours after inoculation.

  These results indicated that these serum neutralizing antibodies showed broader inhibition, as analyzed by pseudovirus inhibition analysis. Overall, these results were consistent with the results obtained from neutralization analysis with wild-type MERS-HCoV, and further showed the cross-protection of the antibody response induced with this vaccine.

Example 16
Multifunctional T cell production in peripheral blood of monkeys after MERS-Spike DNA vaccination Vaccine efficacy against MERS challenge was also evaluated in a preclinical non-human primate model. The study is summarized in Table 2 below.

  Three groups (low, high, and control) of animals (4 Indian rhesus monkeys per group) were immunized with the MERS-Spike vaccine as described in Example 9 (FIG. 23A). Vaccinated animals secrete IFN-γ in response to stimulation with overlapping peptide pools derived from full-length MERS-Spike protein using ELISpot analysis to measure the effect of MERS-Spike vaccine on T cell responses T cells in the blood were listed. After 3 immunizations, the number of MERS-Spike specific T cells present in the blood of the vaccinated animals and the total vaccine response in the low dose group was 600-1100 SFU / 1 million PBMC On the other hand, the high dose group showed 100-1500 SFU / million PBMC except one, and most vaccinated animals had a positive IFN-γ response. Results are shown as stacked group mean response ± standard error (FIGS. 23B and 23C). These data indicated that the MERS-Spike vaccine induced a multifunctional specific T cell response compared to animals that did not receive the vaccine (ie, pVax1 control group).

Example 17
Detection of Humoral Immune Response MERS-spike specific antibodies were detected in sera obtained from vaccinated animals 2 weeks after the third immunization. First, the MERS-Spike specific ELISA was performed using the full length MERS-Spike protein. The results of the binding ELISA are shown in FIGS. 24A, 27A, and 27B. All sera before vaccination (day 0) were negative for MERS-Spike specific antibodies by ELISA. After the third immunization with MERS-Spike, the low dose and high dose groups showed that high titer antibodies were produced. Endpoint MERS-spike specific antibody titers greater than 10,000 were observed in both low and high dose vaccine immunized monkeys with MERS-Spike protein (FIG. 24B). Thus, animals receiving the vaccine have a MERS-Spike antigen-specific humoral immune response, unlike animals that did not receive the vaccine (ie, pVax1 control group).

Example 18
Increased ability to react cross-neutralizing antibodies in non-human primates (NHP) Neutralizing antibodies (Nab) against laboratory-matched stocks of MERS-HCoV were also measured from both pre-bleeds and after the third immunization. Monkeys were immunized three times by IM-EP with either 500 ug or 2 mg of total DNA and showed high concentrations of Nab against live MERS-EMC / 2012 isolate (branch group A) (FIG. 24C). The MERS-CoV genome was phylogenetically divided into two bifurcation groups, bifurcation groups A and B. To analyze this cross-branching group neutralizing activity, cross-neutralization was performed with the MERS pseudovirus that expressed multiple branch groups of Spike protein and induced neutralizing activity against all other MERS-CoV branch groups. Performed (FIG. 24D). Thus, vaccination with DNA expressing the consensus MERS-Spike led to the development of higher antibody titers and more durable Nab responses to MERS-CoV. At the same time, these findings indicated that the MERS-Spike DNA vaccination produced a polyclonal antibody that not only binds to the MERS-Spike protein but is also functionally active and elicited a MERS neutralizing antibody.

Example 19
MERS challenge and pathology after vaccination Experiments were designed to evaluate clinical observations of MERS-CoV inoculated rhesus monkeys.

  As shown in Table 2 above, NHP was challenged intranasally (in) with MERS at week 10 and analyzed at week 12. This 12 week analysis included x-ray data / pathology analysis.

The control group vaccinated with the pVax1 DNA vector exhibited gross lesions throughout the lung lobe and significant lesions in the lower lobe 5 days after challenge. This global lesion in the control group was commensurate with MERS infection. Table 3 summarizes the animal lesions. In summary, the animals vaccinated with the MERS-Spike DNA show improved clinical indicators without gross lesions compared to the animals in the control group, so the MERS-Spike DNA vaccine is Showed that it brought defenses against.
Table 3: Radiographic findings of 1-6 dpi in the lungs of rhesus monkeys inoculated with MERS-CoV. Imaging and clinical observations were performed on days 1, 3, 5, and 6.

6). Article 1. A vaccine comprising a nucleic acid molecule, wherein: (a) said nucleic acid molecule comprises a nucleic acid sequence that is at least about 90% homologous to the full length of the nucleic acid sequence set forth in SEQ ID NO: 1; or (b) said nucleic acid molecule comprises a sequence A vaccine comprising a nucleic acid sequence that is at least about 90% homologous to the full length of the nucleic acid sequence of number 3.

  2. The vaccine of claim 1, wherein the nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 1.

  3. The vaccine of claim 1, wherein the nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 3.

  4). The vaccine according to item 1, further comprising (a) a peptide comprising an amino acid sequence at least about 90% homologous to the full length of the amino acid sequence described in SEQ ID NO: 2, or (b) the amino acid described in SEQ ID NO: 4 The vaccine comprising a peptide comprising an amino acid sequence at least about 90% homologous to the full length of the sequence.

  5. The vaccine of claim 1, wherein the nucleic acid molecule comprises an expression vector.

  6). The vaccine according to item 1, wherein the nucleic acid molecule is incorporated into a virus particle.

  7). 2. The vaccine according to item 1, further comprising a pharmaceutically acceptable excipient.

  8). 2. The vaccine according to item 1, further comprising an adjuvant.

  9. A vaccine comprising a nucleic acid molecule, wherein (a) the nucleic acid molecule encodes a peptide comprising an amino acid sequence that is at least about 90% homologous to the full length of the amino acid sequence set forth in SEQ ID NO: 2, or (b) the nucleic acid A vaccine wherein the molecule encodes a peptide comprising an amino acid sequence that is at least about 90% homologous to the full length of the amino acid sequence set forth in SEQ ID NO: 4.

  10. 10. The vaccine according to item 9 above, wherein the nucleic acid molecule encodes the peptide comprising the amino acid sequence set forth in SEQ ID NO: 2.

  11. 10. The vaccine according to item 9 above, wherein the nucleic acid molecule encodes the peptide comprising the amino acid sequence set forth in SEQ ID NO: 4.

  12 The vaccine according to item 9, further comprising (a) a peptide comprising an amino acid sequence at least about 90% homologous to the full length of the amino acid sequence described in SEQ ID NO: 2, or (b) the amino acid described in SEQ ID NO: 4 The vaccine comprising a peptide comprising an amino acid sequence at least about 90% homologous to the full length of the sequence.

  13. The vaccine according to item 9 above, wherein the nucleic acid molecule comprises an expression vector.

  14 The vaccine according to item 9 above, wherein the nucleic acid molecule is incorporated into a virus particle.

  15. 10. The vaccine according to item 9 further comprising a pharmaceutically acceptable excipient.

  16. 10. The vaccine according to item 9, further comprising an adjuvant.

  17. A nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 1.

  18. A nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 3.

  19. A peptide comprising the amino acid sequence set forth in SEQ ID NO: 2.

  20. A peptide comprising the amino acid sequence set forth in SEQ ID NO: 4.

  21. A vaccine comprising an antigen, wherein the antigen is encoded by SEQ ID NO: 1 or SEQ ID NO: 3.

  22. The vaccine according to item 21, wherein the antigen is encoded by SEQ ID NO: 1.

  23. The vaccine of claim 21, wherein the antigen is encoded by SEQ ID NO: 3.

  24. The vaccine according to item 21 above, wherein the antigen comprises the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4.

  25. 25. The vaccine according to item 24, wherein the antigen comprises the amino acid sequence set forth in SEQ ID NO: 2.

  26. 25. The vaccine according to item 24, wherein the antigen comprises the amino acid sequence set forth in SEQ ID NO: 4.

  27. A vaccine comprising a peptide, wherein (a) the peptide comprises an amino acid sequence that is at least about 90% homologous to the full length of the amino acid sequence set forth in SEQ ID NO: 2, or (b) the peptide comprises SEQ ID NO: 4 A vaccine comprising an amino acid sequence that is at least about 90% homologous to the full length of the described amino acid sequence.

  28. 28. The vaccine according to item 27, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 2.

  29. 28. The vaccine according to item 27 above, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 4.

  30. 28. A method of inducing an immune response against a Middle Eastern respiratory syndrome coronavirus (MERS-CoV) in a patient in need thereof, wherein the method is directed to the patient according to paragraph 1, 9, 21, or 27. A method comprising the administration of

  31. 31. The method of paragraph 30, wherein the administration comprises at least one of electroporation and infusion.

  32. 28. A method of protecting a patient in need of a method of protection from infection by the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), wherein the method comprises the vaccine of claim 1, 9, 21, or 27 to the patient. A method comprising the administration of

  33. 34. The method of paragraph 32, wherein administering comprises at least one of electroporation and infusion.

  34. 28. A method of treating a patient in need of treatment for Middle East Respiratory Syndrome Coronavirus (MERS-CoV), said method comprising administering to said patient the vaccine of paragraph 1, 9, 21, or 27 above And a method whereby the patient is thereby resistant to one or more MERS-CoV strains.

  35. 35. The method of paragraph 34, wherein administering comprises at least one of electroporation and infusion.

  The foregoing detailed description and the accompanying examples are illustrative only and are not intended to limit the scope of the invention, which is defined only by the appended claims and their equivalents. Understood.

  Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention including, without limitation, those relating to the chemical structures, substituents, derivatives, intermediates, synthesis, compositions, formulations, or methods of use of the present invention. Also good.

Claims (35)

An immunogenic composition comprising a nucleic acid molecule,
(A) the nucleic acid molecule comprises a nucleic acid sequence that is at least about 90% homologous to the full length of the nucleic acid sequence set forth in SEQ ID NO: 1; or
(B) An immunogenic composition wherein the nucleic acid molecule comprises a nucleic acid sequence that is at least about 90% homologous to the full length of the nucleic acid sequence set forth in SEQ ID NO: 3.   The immunogenic composition of claim 1, wherein the nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 1.   The immunogenic composition of claim 1, wherein the nucleic acid molecule comprises the nucleic acid sequence set forth in SEQ ID NO: 3. The immunogenic composition of claim 1 further comprising:
(A) a peptide comprising an amino acid sequence at least about 90% homologous to the full length of the amino acid sequence set forth in SEQ ID NO: 2, or
(B) the immunogenic composition comprising a peptide comprising an amino acid sequence at least about 90% homologous to the full length of the amino acid sequence set forth in SEQ ID NO: 4.   2. The immunogenic composition of claim 1 wherein the nucleic acid molecule comprises an expression vector.   The immunogenic composition of claim 1 wherein the nucleic acid molecule is incorporated into a viral particle.   The immunogenic composition of claim 1 further comprising a pharmaceutically acceptable excipient.   The immunogenic composition of claim 1 further comprising an adjuvant. An immunogenic composition comprising a nucleic acid molecule,
(A) the nucleic acid molecule encodes a peptide comprising an amino acid sequence that is at least about 90% homologous to the full length of the amino acid sequence set forth in SEQ ID NO: 2, or
(B) An immunogenic composition wherein the nucleic acid molecule encodes a peptide comprising an amino acid sequence that is at least about 90% homologous to the full length of the amino acid sequence set forth in SEQ ID NO: 4.   The immunogenic composition of claim 9, wherein the nucleic acid molecule encodes the peptide comprising the amino acid sequence set forth in SEQ ID NO: 2.   The immunogenic composition of claim 9, wherein the nucleic acid molecule encodes the peptide comprising the amino acid sequence set forth in SEQ ID NO: 4. An immunogenic composition according to claim 9,
(A) a peptide comprising an amino acid sequence at least about 90% homologous to the full length of the amino acid sequence set forth in SEQ ID NO: 2, or
(B) the immunogenic composition further comprising a peptide comprising an amino acid sequence at least about 90% homologous to the full length of the amino acid sequence set forth in SEQ ID NO: 4.   The immunogenic composition of claim 9, wherein the nucleic acid molecule comprises an expression vector.   The immunogenic composition of claim 9, wherein the nucleic acid molecule is incorporated into a viral particle.   The immunogenic composition of claim 9 further comprising a pharmaceutically acceptable excipient.   The immunogenic composition of claim 9 further comprising an adjuvant.   A nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 1.   A nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 3.   A peptide comprising the amino acid sequence set forth in SEQ ID NO: 2.   A peptide comprising the amino acid sequence set forth in SEQ ID NO: 4.   An immunogenic composition comprising an antigen, wherein said antigen is encoded by SEQ ID NO: 1 or SEQ ID NO: 3.   The immunogenic composition of claim 21, wherein the antigen is encoded by SEQ ID NO: 1.   The immunogenic composition of claim 21, wherein the antigen is encoded by SEQ ID NO: 3.   The immunogenic composition of claim 21, wherein the antigen comprises the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4.   25. The immunogenic composition of claim 24, wherein the antigen comprises the amino acid sequence set forth in SEQ ID NO: 2.   25. The immunogenic composition of claim 24, wherein the antigen comprises the amino acid sequence set forth in SEQ ID NO: 4. An immunogenic composition comprising a peptide comprising:
(A) the peptide comprises an amino acid sequence that is at least about 90% homologous to the full length of the amino acid sequence set forth in SEQ ID NO: 2, or
(B) An immunogenic composition wherein the peptide comprises an amino acid sequence that is at least about 90% homologous to the full length of the amino acid sequence set forth in SEQ ID NO: 4.   28. The immunogenic composition of claim 27, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 2.   28. The immunogenic composition of claim 27, wherein the peptide comprises the amino acid sequence set forth in SEQ ID NO: 4.   28. A method of inducing an immune response against a Middle East respiratory syndrome coronavirus (MERS-CoV) in a patient in need thereof, wherein the method is directed to the patient according to claim 1, 9, 21, or 27. A method comprising the administration of an immunogenic composition.   32. The method of claim 30, wherein administering comprises at least one of electroporation and infusion.   28. A method of protecting a patient in need of protection from infection by a Middle Eastern respiratory syndrome coronavirus (MERS-CoV), the method comprising immunizing the patient according to claim 1, 9, 21, or 27. A method comprising administration of a protogenic composition.   35. The method of claim 32, wherein administering comprises at least one of electroporation and infusion.   28. A method of treating a patient in need of treatment for Middle East Respiratory Syndrome Coronavirus (MERS-CoV), wherein the method is an immunogenic composition according to claim 1, 9, 21, or 27. A method wherein the patient is resistant to one or more MERS-CoV strains.   35. The method of claim 34, wherein administering comprises at least one of electroporation and infusion.